Novel polypeptides

ABSTRACT

The present invention provides a bispecific polypeptides, such as bispecific antibodies, comprising a first binding domain capable of specifically binding to a first T cell target, and a second binding domain capable of specifically binding to a second T cell target, wherein the first and second T cell targets are different targets. The invention further provides compositions of said bispecific polypeptides, as well as methods and uses of the same.

This application is a continuation application of U.S. patentapplication Ser. No. 15/567,163, filed Oct. 17, 2017, which is a § 371application of PCT/EP2016/061420, filed May 20, 2016, which claimspriority to GB Application No. 1508729.9, filed May 21, 2015, GBApplication No. 1514994.1, filed Aug. 24, 2015, and GB Application No.1605450.4, filed Mar. 31, 2016. The entire disclosure of each of theforegoing applications is incorporated by reference herein.

Incorporated herein by reference in its entirety is the Sequence Listingbeing concurrently submitted via EFS-Web as a text file namedSeqList.txt, created Feb. 3, 2020, and having a size of 274,067 bytes.

FIELD OF INVENTION

The present invention relates to bispecific polypeptides whichspecifically bind to two different T cell targets. Exemplary T celltargets include OX40, CTLA-4 and CD137, in particular human OX40, CTLA-4and CD137.

BACKGROUND

Cancer is a leading cause of premature deaths in the developed world.Immunotherapy of cancer aims to mount an effective immune responseagainst tumour cells. This may be achieved by, for example, breakingtolerance against tumour antigen, augmenting anti-tumor immuneresponses, and stimulating local cytokine responses at the tumor site.The key effector cell of a long lasting anti-tumor immune response isthe activated tumor specific effector T cell. Potent expansion ofactivated effector T cells can redirect the immune response towards thetumor. In this context, regulatory T cells (Treg) play a role ininhibiting the anti-tumor immunity. Depleting, inhibiting, reverting orinactivating Tregs may therefore provide anti-tumor effects and revertthe immune suppression in the tumor microenvironment. Further,incomplete activation of effector T cells by, for example, dendriticcells can cause T cell anergy, which results in an inefficientanti-tumor response, whereas adequate induction by dendritic cells cangenerate a potent expansion of activated effector T cells, redirectingthe immune response towards the tumor. In addition, Natural killer (NK)cells play an important role in tumor immunology by attacking tumorcells with down-regulated human leukocyte antigen (HLA) expression andby inducing antibody dependent cellular cytotoxicity (ADCC). Stimulationof NK cells may thus also reduce tumor growth.

OX40 (otherwise known as CD134 or TNFRSF4) is a member of the TNFRfamily that is expressed mainly on activated T cells (mostly CD4+effector T cells, but also CD8+ effector T-cells and regulatory T cells(Tregs)). In mice the expression is constitutive on Tregs, but not inhumans. OX40 expression typically occurs within 24 hours of activation(T cell receptor engagement) and peaks after 48-72 hours. OX40stimulation is important for the survival and proliferation of activatedT cells. The only known ligand for OX40 is OX40L, which is mainlyexpressed on antigen presenting cells, such as dendritic cells and Bcells, typically following their activation. The net result ofOX40-mediated T cell activation is the induction of a TH1 effector Tcell activation profile and a reduction in the activity and/or numbersof Treg cells e.g. via ADCC or ADCP. Overall these effects maycontribute to anti-tumor immunity. OX40 is overexpressed on regulatory Tcells in many solid tumors, such as melanoma, lung cancer and renalcancer.

OX40 agonist treatment of tumor models in mice has been shown to resultin anti-tumor effects and cure of several different cancer forms,including melanoma, glioma, sarcoma, prostate, colon and renal cancers.The data is consistent with a tumor specific T-cell response, involvingboth CD4+ and CD8+ T cells, similar to the effect seen with CD40 agonisttreatments. Addition of IL-12 and other cytokines, and combination withother immunomodulators and chemo/radiotherapy, has been shown to improvethe therapeutic effect of OX40 agonist treatment. Evidence frompre-clinical models suggests that the effect of anti-OX40 antibodies isdependent upon activating FcγR. A clinical phase I study testing themouse anti-human OX40 Clone 9B12 in late stage patients that had failedall other therapy has been conducted at the Providence Cancer Centre.The antibody was well-tolerated. Tumor shrinkage and an increase in CD4+and CD8+ T cell proliferation were observed. The low toxicity may becaused by low half-life and anti-drug antibodies (the antibody was amouse antibody), but also by the relatively low expression levels ofOX40 on non-activated T cells. The anti-tumor effect with this antibodywas modest.

CD137 (4-1BB, TNFRSF9) is also a member of the TNFR family. Activationof CD137 is dependent on receptor oligomerization. CD137 is expressed onactivated CD4+ and CD8+ T cells, Treg, DC, monocytes, mast cells andeosinophils. CD137 activation plays an important role in CD8+ T cellactivation and survival. It sustains and augments, rather than initiateseffector functions and preferentially supports TH1 cytokine production.In CD4+ T cells, CD137 stimulation initially results in activation andlater in activation-induced cell death, which may explain why CD137agonistic antibodies have shown therapeutic effect in tumor immunity aswell as in autoimmunity. CD137 also suppresses Treg function. CD137 isupregulated on NK cells activated by cytokines or CD16. Activation ofCD137 on NK cells has been shown to increase ADCC activity of NK cellsin both murine and human cells. Further, CD137 is expressed on antigenpresenting cells, such as dendritic cells and macrophages, andstimulation of CD137 on these cell types may induce immune activationthat can result in tumor directed immunity. CD137 agonistic antibody hasalso been shown to activate endothelial cells in the tumor environment,leading to upregulation of ICAM-1 and VCAM-1 and improved T cellrecruitment. Several studies have demonstrated induction of tumorimmunity by treatment with agonistic CD137 antibodies.

Two CD137 antibodies are in clinical development. Urelumab (BMS-66513)is a fully human IgG4 antibody developed by Bristol-Myers Squibb.Several phase I and II studies in various indications are currentlyongoing. A Phase II study with Urelumab as a second line therapy inmetastatic melanoma was terminated in 2009 due to fatal hepatoxicity.The other CD137 mAb in clinical development is PF-05082566, a fullyhuman IgG2 antibody developed by Pfizer. It is currently in phase Idevelopment in lymphoma and various solid cancers and preliminary datasuggest that it is well tolerated but with only modest anti-tumoreffects.

Existing antibodies targeting CD137 or OX40 are in general dependent oncross linking via e.g. Fcgamma Receptors on other cells to induce strongsignaling into cells expressing the respective receptor. Thus, they donot signal efficiently when no such cross linking is provided. Inaddition, prolonged and continuous activation through TNF receptorfamily members may lead to immune exhaustion.

The T cell receptor CTLA-4, serves as a negative regulator of T cellactivation, and is upregulated on the T-cell surface following initialactivation. The ligands of the CTLA-4 receptor, which are expressed byantigen presenting cells are the B7 proteins. The corresponding ligandreceptor pair that is responsible for the upregulation of T cellactivation is CD28-B7. Signalling via CD28 constitutes a costimulatorypathway, and follows upon the activation of T cells, through the T cellreceptor recognizing antigenic peptide presented by the MHC complex. Byblocking the CTLA-4 interaction to the B7-1 and, or B7-2 ligands, one ofthe normal check points of the immune response may be removed. The netresult is enhanced activity of effector T cells which may contribute toanti-tumour immunity. As with OX40, this may be due to direct activationof the effector T cells but may also be due to a reduction in theactivity and/or numbers of Treg cells, e.g. via ADCC or ADCP. Clinicalstudies have demonstrated that CTLA-4 blockade generates anti-tumoreffects, but administration of anti-CTLA-4 antibodies has beenassociated with toxic side-effects. CTLA-4 is overexpressed onregulatory T cells in many solid tumors, such as melanoma lung cancerand renal cancer.

There is a need for an alternative to the existing monospecific drugsthat target only one T cell target, such as OX40 or CD137 or CTLA-4.

SUMMARY OF INVENTION

A first aspect of the invention provides a bispecific polypeptidecomprising a first binding domain, designated B1, which is capable ofspecifically binding to a first T cell target, and a second bindingdomain, designated B2, which is capable of specifically binding to asecond T cell target, wherein the first and second T cell targets aredifferent targets.

Bispecific antibodies targeting two different T cell targets, such asCTLA-4, CD137 and OX40, have the potential to specifically activate theimmune system in locations were both targets are over expressed. Forexample, CTLA-4 and OX40 are overexpressed on regulatory T cells (Treg)in the tumor microenvironment, whereas their expression on effector Tcells is lower. Thus, the bispecific antibodies of the invention havethe potential to selectively target regulatory T cells in the tumormicroenvironment.

Targeting Treg cells in the tumor microenvironment with a bispecificantibody of the invention also has the potential to deplete or reversethe immune suppressive function of the Tregs. This effect could bemediated by ADCC or ADCP induction via the Fc part of the bispecificantibody of the invention (for example, see Furness et al., 2014 TrendsImmunol 35(7):290-8; the disclosures of which are incorporated herein byreference) or by signaling induced via OX40 and/or CTLA-4 and/or byblocking the CTLA-4 signaling pathway (for example, see Walker, 2014,Nature Reviews 11(12):852-63; the disclosures of which are incorporatedherein by reference). On effector T cells, on the other hands, thebispecific antibodies of the invention have the potential to induceactivation and increased function both via OX40 stimulation and throughCTLA-4 checkpoint blockade.

The net effects of the bispecific antibodies targeting two T-cellreceptors are thus:

-   -   1. A higher degree of immune activation compared to monospecific        antibodies. The immune activation is higher than the combination        of the monospecific antibodies.    -   2. A higher degree of induction of ADCC compared to the        monospecific binders in combination.    -   3. A more directed/localized immune activation. The immune        activation only occurs in environments that contains both high        CTLA-4 expression and OX40 expression. The tumor        microenvironment is such an environment. This has the potential        to increase the effect and also to minimize toxic side effect.        Thus the therapeutic window will be increased.

In exemplary embodiments, the bispecific polypeptide is capable ofbinding specifically to:

-   -   (a) OX40 and CTLA-4;    -   (b) OX40 and CD137; or    -   (c) CD137 and CTLA-4.

A “polypeptide” is used herein in its broadest sense to refer to acompound of two or more subunit amino acids, amino acid analogs, orother peptidomimetics. The term “polypeptide” thus includes shortpeptide sequences and also longer polypeptides and proteins. As usedherein, the term “amino acid” refers to either natural and/or unnaturalor synthetic amino acids, including both D or L optical isomers, andamino acid analogs and peptidomimetics.

The term “bispecific” as used herein means the polypeptide is capable ofspecifically binding at least two target entities.

In one embodiment the first and/or second binding domains may beselected from the group consisting of: antibodies or antigen-bindingfragments thereof.

For example, the bispecific polypeptide of the invention may comprise:

-   -   (i) a first binding domain which comprises or consists of an        antibody variable domain or part thereof and a second binding        domain which comprises or consists of an antibody variable        domain or part thereof; or    -   (ii) a first binding domain which comprises or consists of an        antibody variable domain or part thereof and a second binding        domain which is not an antibody variable domain or part thereof.

Thus, in one embodiment the polypeptide is a bispecific antibody.

As used herein, the terms “antibody” or “antibodies” refer to moleculesthat contain an antigen binding site, e.g. immunoglobulin molecules andimmunologically active fragments of immunoglobulin molecules thatcontain an antigen binding site. Immunoglobulin molecules can be of anytype (e.g. IgG, IgE, IgM, IgD, IgA and IgY), class (e.g. IgG1, IgG2,IgG3, IgG4, IgA1 and IgA2) or a subclass of immunoglobulin molecule.Antibodies include, but are not limited to, synthetic antibodies,monoclonal antibodies, single domain antibodies, single chainantibodies, recombinantly produced antibodies, multi-specific antibodies(including bi-specific antibodies), human antibodies, humanizedantibodies, chimeric antibodies, intrabodies, scFvs (e.g. includingmono-specific and bi-specific, etc.), Fab fragments, F(ab′) fragments,disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies, andepitope-binding fragments of any of the above.

The terms antibody “directed to” or “directed against” are usedinterchangeably herein and refer to an antibody that is constructed todirect its binding specificity(ies) at a certaintarget/marker/epitope/antigen, i.e. an antibody that immunospecificallybinds to a target/marker/epitope/antigen. Also, the expressionantibodies “selective for” a certain target/marker/epitope may be used,having the same definition as “directed to” or “directed against”. Abi-specific antibody directed to (selective for) two differenttargets/markers/epitopes/antigens binds immunospecifically to bothtargets/markers/epitopes/antigens.

If an antibody is directed to a certain target antigen, such as CD137,it is thus assumed that said antibody could be directed to any suitableepitope present on said target antigen structure.

As used herein, the term “antibody fragment” is a portion of an antibodysuch as F(ab′).sub.2, F(ab).sub.2, Fab′, Fab, Fv, scFv and the like.Regardless of structure, an antibody fragment binds with the sameantigen that is recognized by the intact antibody. For example, ananti-OX4 antibody fragment binds to OX40. The term “antibody fragment”also includes isolated fragments consisting of the variable regions,such as the “Fv” fragments consisting of the variable regions of theheavy and light chains and recombinant single chain polypeptidemolecules in which light and heavy variable regions are connected by apeptide linker (“scFv proteins”). As used herein, the term “antibodyfragment” does not include portions of antibodies without antigenbinding activity, such as Fc fragments or single amino acid residues.

ScFv are particularly preferred for inclusion in the bispecificantibodies of the invention.

Thus, in exemplary embodiments of the bispecific antibodies of theinvention:

-   -   (a) binding domain B1 and/or binding domain B2 is an intact IgG        antibody (or, together, form an intact IgG antibody);    -   (b) binding domain B1 and/or binding domain B2 is an Fv fragment        (e.g. an scFv);    -   (c) binding domain B1 and/or binding domain B2 is a Fab        fragment; and/or    -   (d) binding domain B1 and/or binding domain B2 is a single        domain antibody (e.g. domain antibodies and nanobodies).

It will be appreciated by persons skilled in the art that the bispecificpolypeptides of the invention may be of several different structuralformats (for example, see Chan & Carter, 2016, Nature Reviews Immunology10, 301-316, the disclosures of which are incorporated herein byreference).

In exemplary embodiments, the bispecific antibody is selected from thegroups consisting of:

-   -   (a) bivalent bispecific antibodies, such as IgG-scFv bispecific        antibodies (for example, wherein B1 is an intact IgG and B2 is        an scFv attached to B1 at the N-terminus of a light chain and/or        at the C-terminus of a light chain and/or at the N-terminus of a        heavy chain and/or at the C-terminus of a heavy chain of the        IgG, or vice versa);    -   (b) monovalent bispecific antibodies, such as a DuoBody® (Genmab        AS, Copenhagen, Denmark) or ‘knob-in-hole’ bispecific antibody        (for example, an scFv-KIH, scFv-KIH^(r), a BiTE-KIH or a        BiTE-KIH^(r) (see Xu et al., 2015, mAbs 7(1):231-242);    -   (c) scFv₂-Fc bispecific antibodies (such as ADAPTIR™ bispecific        antibodies from Emergent Biosolutions Inc);    -   (d) BiTE/scFv₂ bispecific antibodies;    -   (e) DVD-Ig bispecific antibodies;    -   (f) DART-based bispecific antibodies (for example, DART₂-Fc,        DART₂-Fc or DART);    -   (g) DNL-Fab₃ bispecific antibodies; and    -   (h) scFv-HSA-scFv bispecific antibodies.

For example, the bispecific antibody may be an IgG-scFv antibody. TheIgG-scFv antibody may be in either VH-VL or VL-VH orientation. In oneembodiment, the scFv may be stabilised by a S—S bridge between VH andVL.

In one embodiment, binding domain B1 and binding domain B2 are fuseddirectly to each other.

In an alternative embodiment, binding domain B1 and binding domain B2are joined via a polypeptide linker. For example, a polypeptide linkermay be a short linker peptide between about 10 to about 25 amino acids.The linker is usually rich in glycine for flexibility, as well as serineor threonine for solubility, and can either connect the N-terminus ofthe VH with the C-terminus of the VL, or vice versa. Exemplary linkersinclude a peptide of amino acid sequence as shown in any one of SEQ IDNOs. 47 to 50, or 144. The peptide of sequence GGGGSGGGGSGGGGS (SEQ IDNO: 144) is particularly preferred. The same linkers may be used to jointhe anti-OX40 part of a bispecific antibody of the invention to theanti-CD137 part.

The bispecific polypeptides of the invention may be manufactured by anyknown suitable method used in the art. Methods of preparing bi-specificantibodies of the present invention include BiTE (Micromet), DART(MacroGenics), Fcab and Mab² (F-star), Fc-engineered IgGI (Xencor) orDuoBody (based on Fab arm exchange, Genmab). Examples of other platformsuseful for preparing bi-specific antibodies include but are not limitedto those described in WO 2008/119353 (Genmab), WO 2011/131746 (Genmab)and reported by van der Neut-Kolfschoten et al. (2007, Science317(5844):1554-7). Traditional methods such as the hybrid hybridoma andchemical conjugation methods (Marvin and Zhu (2005) Acta Pharmacol Sin26: 649) can also be used. Co-expression in a host cell of twoantibodies, consisting of different heavy and light chains, leads to amixture of possible antibody products in addition to the desiredbi-specific antibody, which can then be isolated by, e.g. affinitychromatography or similar methods.

It will be appreciated by persons skilled in the art that the bispecificantibody may comprise a human Fc region, or a variant of a said region,where the region is an IgG1, IgG2, IgG3 or IgG4 region, preferably anIgG1 or IgG4 region.

The constant (Fc) regions of the antibodies may mediate the binding ofthe immunoglobulin to host tissues or factors, including various cellsof the immune system (e.g., effector cells) and the first component(Clq) of the classical complement system. The Fc region is preferably ahuman Fc region, or a variant of a said region. The Fc region may be anIgG1, IgG2, IgG3 or IgG4 region, preferably an IgG1 or IgG4 region. Avariant of an Fc region typically binds to Fc receptors, such asFcgammaR and/or neonatal Fc receptor (FcRn) with altered affinityproviding for improved function and/or half-life of the polypeptide. Thebiological function and/or the half-life may be either increased or adecreased relative to the half-life of a polypeptide comprising a nativeFc region. Examples of such biological functions which may be modulatedby the presence of a variant Fc region include antibody dependent cellcytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP),complement-dependent cytotoxicity (CDC), and/or apoptosis.

An exemplary heavy chain constant region amino acid sequence which maybe combined with any VH region sequence disclosed herein (to form acomplete heavy chain) is the IgG1 heavy chain constant region sequencereproduced here:

(SEQ ID NO: 135) ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

Other heavy chain constant region sequences are known in the art andcould also be combined with any VH region disclosed herein. For example,a preferred constant region is a modified IgG4 constant region such asthat reproduced here:

(SEQ ID NO: 137) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNRYTQKSLSLSLGK

This modified IgG4 sequence exhibits reduced FcRn binding and henceresults in a reduced serum half-life relative to wild type IgG4. Inaddition, it exhibits stabilization of the core hinge of IgG4 making theIgG4 more stable, preventing Fab arm exchange.

Another preferred constant region is a modified IgG4 constant regionsuch as that reproduced here:

(SEQ ID NO: 139) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

This modified IgG4 sequence results in stabilization of the core hingeof IgG4 making the IgG4 more stable, preventing Fab arm exchange

Also preferred is a wild type IgG4 constant region such as thatreproduced here:

(SEQ ID NO: 138) ASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

An exemplary light chain constant region amino acid sequence which maybe combined with any VL region sequence disclosed herein (to form acomplete light chain) is the kappa chain constant region sequencereproduced here:

(SEQ ID NO: 136) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC

Other light chain constant region sequences are known in the art andcould also be combined with any VL region disclosed herein.

The antibody, or antigen binding fragment thereof, has certain preferredbinding characteristics and functional effects, which are explained inmore detail below. Said antibody, or antigen binding fragment thereof,preferably retains these binding characteristics and functional effectswhen incorporated as part of a bispecific polypeptide of the invention.

In one embodiment the antigen-binding fragment may be selected from thegroup consisting of: an Fv fragment (such as a single chain Fv fragment,or a disulphide-bonded Fv fragment), a Fab-like fragment (such as a Fabfragment; a Fab′ fragment or a F(ab)₂ fragment) and domain antibodies.

In one embodiment the bispecific polypeptide may be an IgG1 antibodywith a non-immunoglobulin polypeptide (such as a CTLA-4 binding domain,e.g. CD86 or a mutated form thereof such as SEQ ID NO: 17; see below)fused to the C-terminal part of the kappa chain.

In one embodiment the bispecific polypeptide may be an IgG1 antibodywith a scFv fragment fused to the C-terminal end of the heavy gamma 1chain.

In one embodiment the bispecific polypeptide may contain 2-4 scFvbinding to two different targets.

By “T cell target” we include polypeptide receptors located in the cellmembrane of CD3+ T cells in an activated or inactive state. Suchmembrane-bound receptors may be exposed extracellularly in order thatthey accessed by the bispecific polypeptides of the invention followingadministration.

It will be appreciated by persons skilled in the art that the T celltargets may be localised on the surface of a cell. By “localised on thesurface of a cell” it is meant that the T cell target is associated withthe cell such that one or more region of the T cell target is present onthe outer face of the cell surface. For example, the T cell target maybe inserted into the cell plasma membrane (i.e. orientated as atransmembrane protein) with one or more regions presented on theextracellular surface. This may occur in the course of expression of theT cell target by the cell. Thus, in one embodiment, “localised on thesurface of a cell” may mean “expressed on the surface of a cell.”Alternatively, the T cell target may be outside the cell with covalentand/or ionic interactions localising it to a specific region or regionsof the cell surface.

In one embodiment the first and/or second T cell target may be acheckpoint molecule. For example, in one embodiment the first and/orsecond T cell target is a co-stimulatory or co-inhibitory molecule.

By “co-stimulatory” we include co-signalling molecules which are capableof promoting T cell activation. By “co-inhibitory” we includeco-signalling molecules which are capable of suppressing T cellactivation.

Accordingly, in one embodiment at least one of the T cell targets may bea stimulatory checkpoint molecule (such as CD137, GITR, CD27, CD28, ICOSand OX40).

Advantageously, the bispecific polypeptide of the invention is anagonist at a stimulatory checkpoint molecule.

Alternatively or additionally, at least one of the T cell targets may bean inhibitory checkpoint molecule (such as CTLA-4, PD-1, Tim3, Lag3,Tigit or VISTA).

Advantageously, the bispecific polypeptide of the invention is anantagonist at an inhibitory checkpoint molecule.

In one embodiment at least one of the T cell targets is a TNFR (tumornecrosis factor receptor) superfamily member. By TNFR superfamily memberwe include cytokine receptors characterised by the ability to bind tumornecrosis factors (TNFs) via an extracellular cysteine-rich domain.Examples of TNFRs include OX40 and CD137.

In one embodiment the first and/or second T cell target may be selectedfrom the group consisting of: OX40, CTLA-4, CD137, CD40 and CD28. Forexample, the first and/or second T cell target may be selected from thegroup consisting of OX40, CTLA-4 and CD137.

It will be appreciated by persons skilled in the art that the bispecificantibodies of the invention may be capable of inducing antibodydependent cell cytotoxicity (ADCC), antibody-dependent cellularphagocytosis (ADCP), complement-dependent cytotoxicity (CDC), and/orapoptosis.

In a further embodiment the polypeptide is capable of inducing tumourimmunity. This can be tested in vitro in T cell activation assays, e.g.by measuring. IL-2 and IFNγ production. Activation of effector T cellswould imply that a tumor specific T cell response can be achieved invivo. Further, an anti-tumor response in an in vivo model, such as amouse model would imply that a successful immune response towards thetumor has been achieved.

The antibody may modulate the activity of a cell expressing the T celltarget, wherein said modulation is an increase or decrease in theactivity of said cell. The cell is typically a T cell. The antibody mayincrease the activity of a CD4+ or CD8+ effector cell, or may decreasethe activity of a regulatory T cell (Treg). In either case, the neteffect of the antibody will be an increase in the activity of effector Tcells, particularly CD4+ effector T cells. Methods for determining achange in the activity of effector T cells are well known and include,for example, measuring for an increase in the level of T cell IL-2production or an increase in T cell proliferation in the presence of theantibody relative to the level of T cell IL-2 production and/or T cellproliferation in the presence of a control. Assays for cellproliferation and/or IL-2 production are well known and are exemplifiedin the Examples.

Standard assays to evaluate the binding ability of ligands towardstargets are well known in the art, including for example, ELISAs,Western blots, RIAs, and flow cytometry analysis. The binding kinetics(e.g., binding affinity) of the polypeptide also can be assessed bystandard assays known in the art, such as by Surface Plasmon Resonanceanalysis (SPR).

The terms “binding activity” and “binding affinity” are intended torefer to the tendency of a polypeptide molecule to bind or not to bindto a target. Binding affinity may be quantified by determining thedissociation constant (Kd) for a polypeptide and its target. A lower Kdis indicative of a higher affinity for a target. Similarly, thespecificity of binding of a polypeptide to its target may be defined interms of the comparative dissociation constants (Kd) of the polypeptidefor its target as compared to the dissociation constant with respect tothe polypeptide and another, non-target molecule.

The value of this dissociation constant can be determined directly bywell-known methods, and can be computed even for complex mixtures bymethods such as those, for example, set forth in Caceci et al. (Byte9:340-362, 1984; the disclosures of which are incorporated herein byreference). For example, the Kd may be established using a double-filternitrocellulose filter binding assay such as that disclosed by Wong &Lohman (Proc. Natl. Acad. Sci. USA 90, 5428-5432, 1993). Other standardassays to evaluate the binding ability of ligands such as antibodiestowards targets are known in the art, including for example, ELISAs,Western blots, RIAs, and flow cytometry analysis. The binding kinetics(e.g., binding affinity) of the antibody also can be assessed bystandard assays known in the art, such as by Biacore™ system analysis.

A competitive binding assay can be conducted in which the binding of theantibody to the target is compared to the binding of the target byanother, known ligand of that target, such as another antibody. Theconcentration at which 50% inhibition occurs is known as the Ki. Underideal conditions, the Ki is equivalent to Kd. The Ki value will never beless than the Kd, so measurement of Ki can conveniently be substitutedto provide an upper limit for Kd.

Alternative measures of binding affinity include EC50 or IC50. In thiscontext EC50 indicates the concentration at which a polypeptide achieves50% of its maximum binding to a fixed quantity of target. IC50 indicatesthe concentration at which a polypeptide inhibits 50% of the maximumbinding of a fixed quantity of competitor to a fixed quantity of target.

In both cases, a lower level of EC50 or IC50 indicates a higher affinityfor a target. The EC50 and IC50 values of a ligand for its target canboth be determined by well-known methods, for example ELISA. Suitableassays to assess the EC50 and IC50 of polypeptides are set out in theExamples.

A polypeptide of the invention is preferably capable of binding to itstarget with an affinity that is at least two-fold, 10-fold, 50-fold,100-fold or greater than its affinity for binding to another non-targetmolecule.

The polypeptide of the invention may be produced by any suitable means.For example, all or part of the polypeptide may be expressed as a fusionprotein by a cell comprising a nucleotide which encodes saidpolypeptide.

Alternatively parts B1 and B2 may be produced separately and thensubsequently joined together. Joining may be achieved by any suitablemeans, for example using the chemical conjugation methods and linkersoutlined above. Separate production of parts B1 and B2 may be achievedby any suitable means. For example by expression from separatenucleotides optionally in separate cells, as is explained in more detailbelow.

Variants

The bispecific polypeptides or constituent binding domains thereof (suchas the OX40, CD137 and CTLA-4 binding domains) described herein maycomprise a variant or a fragment of any of the specific amino acidsequences recited herein, provided that the polypeptide or bindingdomain retains binding to its target. In one embodiment the variant ofan antibody or antigen binding fragment may retain the CDR sequences ofthe sequences recited herein. For example, the anti-OX40 or anti-CD137antibody may comprise a variant or a fragment of any of the specificamino acid sequences recited in Tables B and H, provided that theantibody retains binding to its target. Such a variant or fragment maytypically retain the CDR sequences of the said sequence of Table B or H.The CTLA-4 binding domain may comprise a variant of any of the sequencesof Table C, providing that that the binding domain retains binding toits target.

A fragment of any one of the heavy or light chain amino acid sequencesrecited herein may comprise at least 7, at least 8, at least 9, at least10, at least 12, at least 15, at least 18, at least 20, at least 25, atleast 50, at least 60, at least 70, at least 80, at least 90 or at least100 consecutive amino acids from the said amino acid sequence.

A variant of any one of the heavy or light chain amino acid sequencesrecited herein may be a substitution, deletion or addition variant ofsaid sequence. A variant may comprise 1, 2, 3, 4, 5, up to 10, up to 20,up to 30 or more amino acid substitutions and/or deletions from the saidsequence. “Deletion” variants may comprise the deletion of individualamino acids, deletion of small groups of amino acids such as 2, 3, 4 or5 amino acids, or deletion of larger amino acid regions, such as thedeletion of specific amino acid domains or other features.“Substitution” variants preferably involve the replacement of one ormore amino acids with the same number of amino acids and makingconservative amino acid substitutions. For example, an amino acid may besubstituted with an alternative amino acid having similar properties,for example, another basic amino acid, another acidic amino acid,another neutral amino acid, another charged amino acid, anotherhydrophilic amino acid, another hydrophobic amino acid, another polaramino acid, another aromatic amino acid or another aliphatic amino acid.Some properties of the 20 main amino acids which can be used to selectsuitable substituents are as follows:

Ala, A aliphatic, hydrophobic, neutral Met, M hydrophobic, neutral Cys,C polar, hydrophobic, neutral Asn, N polar, hydrophilic, neutral Asp, Dpolar, hydrophilic, charged (−) Pro, P hydrophobic, neutral Glu, Epolar, hydrophilic, charged (−) Gln, Q polar, hydrophilic, neutral Phe,F aromatic, hydrophobic, Arg, R polar, hydrophilic, neutral charged (+)Gly, G aliphatic, neutral Ser, S polar, hydrophilic, neutral His, Haromatic, polar, hydrophilic, Thr, T polar, hydrophilic, charged (+)neutral Ile, I aliphatic, hydrophobic, Val, V aliphatic, hydrophobic,neutral neutral Lys, K polar, hydrophilic, Trp, W aromatic, hydrophobic,charged(+) neutral Leu, L aliphatic, hydrophobic, Tyr, Y aromatic,polar, neutral hydrophobic

Amino acids herein may be referred to by full name, three letter code orsingle letter code.

Preferred “derivatives” or “variants” include those in which instead ofthe naturally occurring amino acid the amino acid which appears in thesequence is a structural analog thereof. Amino acids used in thesequences may also be derivatised or modified, e.g. labelled, providingthe function of the antibody is not significantly adversely affected.

Derivatives and variants as described above may be prepared duringsynthesis of the antibody or by post-production modification, or whenthe antibody is in recombinant form using the known techniques ofsite-directed mutagenesis, random mutagenesis, or enzymatic cleavageand/or ligation of nucleic acids.

Preferably variants have an amino acid sequence which has more than 60%,or more than 70%, e.g. 75 or 80%, preferably more than 85%, e.g. morethan 90 or 95% amino acid identity to a sequence as shown in thesequences disclosed herein. This level of amino acid identity may beseen across the full length of the relevant SEQ ID NO sequence or over apart of the sequence, such as across 20, 30, 50, 75, 100, 150, 200 ormore amino acids, depending on the size of the full length polypeptide.

In connection with amino acid sequences, “sequence identity” refers tosequences which have the stated value when assessed using ClustalW(Thompson et al., 1994, Nucleic Acids Res. 22(22):4673-80; thedisclosures of which are incorporated herein by reference) with thefollowing parameters:

Pairwise alignment parameters—Method: accurate, Matrix: PAM, Gap openpenalty: 10.00, Gap extension penalty: 0.10;

Multiple alignment parameters—Matrix: PAM, Gap open penalty: 10.00, %identity for delay: 30, Penalize end gaps: on, Gap separation distance:0, Negative matrix: no, Gap extension penalty: 0.20, Residue-specificgap penalties: on, Hydrophilic gap penalties: on, Hydrophilic residues:GPSNDQEKR. Sequence identity at a particular residue is intended toinclude identical residues which have simply been derivatised.

Polynucleotides, Vectors and Cells

The invention also relates to polynucleotides that encode all or part ofa polypeptide of the invention. Thus, a polynucleotide of the inventionmay encode any polypeptide as described herein, or all or part of B1 orall or part of B2. The terms “nucleic acid molecule” and“polynucleotide” are used interchangeably herein and refer to apolymeric form of nucleotides of any length, either deoxyribonucleotidesor ribonucleotides, or analogs thereof. Non-limiting examples ofpolynucleotides include a gene, a gene fragment, messenger RNA (mRNA),cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA ofany sequence, isolated RNA of any sequence, nucleic acid probes, andprimers.

A polynucleotide of the invention may be provided in isolated orsubstantially isolated form. By substantially isolated, it is meant thatthere may be substantial, but not total, isolation of the polypeptidefrom any surrounding medium. The polynucleotides may be mixed withcarriers or diluents which will not interfere with their intended useand still be regarded as substantially isolated.

A nucleic acid sequence which “encodes” a selected polypeptide is anucleic acid molecule which is transcribed (in the case of DNA) andtranslated (in the case of mRNA) into a polypeptide in vivo when placedunder the control of appropriate regulatory sequences. The boundaries ofthe coding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxy) terminus. Forthe purposes of the invention, such nucleic acid sequences can include,but are not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA,genomic sequences from viral or prokaryotic DNA or RNA, and evensynthetic DNA sequences. A transcription termination sequence may belocated 3′ to the coding sequence.

Representative polynucleotides which encode examples of a heavy chain orlight chain amino acid sequence of an antibody may comprise or consistof any one of the nucleotide sequences disclosed herein, for example thesequences set out in Table B or H. Representative polynucleotides whichencode the polypeptides shown in Tables D, G or

H may comprise or consist of the corresponding nucleotide sequenceswhich are also shown in Tables D, G or H (intron sequences are shown inlower case). Representative polynucleotides which encode examples ofCTLA-4 binding domains may comprise or consist of any one of SEQ ID NOS:25 to 43 as shown in Table E.

A suitable polynucleotide sequence may alternatively be a variant of oneof these specific polynucleotide sequences. For example, a variant maybe a substitution, deletion or addition variant of any of the abovenucleic acid sequences. A variant polynucleotide may comprise 1, 2, 3,4, 5, up to 10, up to 20, up to 30, up to 40, up to 50, up to 75 or morenucleic acid substitutions and/or deletions from the sequences given inthe sequence listing.

Suitable variants may be at least 70% homologous to a polynucleotide ofany one of nucleic acid sequences disclosed herein, preferably at least80 or 90% and more preferably at least 95%, 97% or 99% homologousthereto. Preferably homology and identity at these levels is present atleast with respect to the coding regions of the polynucleotides. Methodsof measuring homology are well known in the art and it will beunderstood by those of skill in the art that in the present context,homology is calculated on the basis of nucleic acid identity. Suchhomology may exist over a region of at least 15, preferably at least 30,for instance at least 40, 60, 100, 200 or more contiguous nucleotides.Such homology may exist over the entire length of the unmodifiedpolynucleotide sequence.

Methods of measuring polynucleotide homology or identity are known inthe art. For example the UWGCG Package provides the BESTFIT programwhich can be used to calculate homology (e.g. used on its defaultsettings) (Devereux et al, 1984, Nucleic Acids Research 12:387-395; thedisclosures of which are incorporated herein by reference).

The PILEUP and BLAST algorithms can also be used to calculate homologyor line up sequences (typically on their default settings), for exampleas described in Altschul, 1993, J Mol Evol 36:290-300; Altschul et al,1990, J Mol Biol 215:403-10, the disclosures of which are incorporatedherein by reference).

Software for performing BLAST analysis is publicly available through theNational Centre for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). This algorithm involves firstidentifying high scoring sequence pair (HSPs) by identifying short wordsof length W in the query sequence that either match or satisfy somepositive-valued threshold score T when aligned with a word of the samelength in a database sequence. T is referred to as the neighbourhoodword score threshold (Altschul et al, supra). These initialneighbourhood word hits act as seeds for initiating searches to findHSPs containing them. The word hits are extended in both directionsalong each sequence for as far as the cumulative alignment score can beincreased. Extensions for the word hits in each direction are haltedwhen:

the cumulative alignment score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, Tand X determine the sensitivity and speed of the alignment. The BLASTprogram uses as defaults a word length (W) of 11, the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, 1992, Proc. Natl. Acad. Sci. USA89:10915-10919; the disclosures of which are incorporated herein byreference) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and acomparison of both strands.

The BLAST algorithm performs a statistical analysis of the similaritybetween two sequences; see e.g. Karlin & Altschul, 1993, Proc. Natl.Acad. Sci. USA 90:5873-5787; the disclosures of which are incorporatedherein by reference. One measure of similarity provided by the BLASTalgorithm is the smallest sum probability (P(N)), which provides anindication of the probability by which a match between two nucleotide oramino acid sequences would occur by chance. For example, a sequence isconsidered similar to another sequence if the smallest sum probabilityin comparison of the first sequence to the second sequence is less thanabout 1, preferably less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

The homologue may differ from a sequence in the relevant polynucleotideby less than 3, 5, 10, 15, 20 or more mutations (each of which may be asubstitution, deletion or insertion). These mutations may be measuredover a region of at least 30, for instance at least 40, 60 or 100 ormore contiguous nucleotides of the homologue.

In one embodiment, a variant sequence may vary from the specificsequences given in the sequence listing by virtue of the redundancy inthe genetic code. The DNA code has 4 primary nucleic acid residues (A,T, C and G) and uses these to “spell” three letter codons whichrepresent the amino acids the proteins encoded in an organism's genes.The linear sequence of codons along the DNA molecule is translated intothe linear sequence of amino acids in the protein(s) encoded by thosegenes. The code is highly degenerate, with 61 codons coding for the 20natural amino acids and 3 codons representing “stop” signals. Thus, mostamino acids are coded for by more than one codon—in fact several arecoded for by four or more different codons. A variant polynucleotide ofthe invention may therefore encode the same polypeptide sequence asanother polynucleotide of the invention, but may have a differentnucleic acid sequence due to the use of different codons to encode thesame amino acids.

A polypeptide of the invention may thus be produced from or delivered inthe form of a polynucleotide which encodes and is capable of expressingit.

Polynucleotides of the invention can be synthesised according to methodswell known in the art, as described by way of example in Green &Sambrook (2012, Molecular Cloning—a laboratory manual, 4th edition; ColdSpring Harbor Press; the disclosures of which are incorporated herein byreference).

The nucleic acid molecules of the present invention may be provided inthe form of an expression cassette which includes control sequencesoperably linked to the inserted sequence, thus allowing for expressionof the polypeptide of the invention in vivo. These expression cassettes,in turn, are typically provided within vectors (e.g., plasmids orrecombinant viral vectors). Such an expression cassette may beadministered directly to a host subject. Alternatively, a vectorcomprising a polynucleotide of the invention may be administered to ahost subject. Preferably the polynucleotide is prepared and/oradministered using a genetic vector. A suitable vector may be any vectorwhich is capable of carrying a sufficient amount of genetic information,and allowing expression of a polypeptide of the invention.

The present invention thus includes expression vectors that comprisesuch polynucleotide sequences. Such expression vectors are routinelyconstructed in the art of molecular biology and may for example involvethe use of plasmid DNA and appropriate initiators, promoters, enhancersand other elements, such as for example polyadenylation signals whichmay be necessary, and which are positioned in the correct orientation,in order to allow for expression of a peptide of the invention. Othersuitable vectors would be apparent to persons skilled in the art (seeGreen & Sambrook, supra).

The invention also includes cells that have been modified to express apolypeptide of the invention. Such cells include transient, orpreferably stable higher eukaryotic cell lines, such as mammalian cellsor insect cells, lower eukaryotic cells, such as yeast or prokaryoticcells such as bacterial cells. Particular examples of cells which may bemodified by insertion of vectors or expression cassettes encoding for apolypeptide of the invention include mammalian HEK293T, CHO, HeLa, NSOand COS cells. Preferably the cell line selected will be one which isnot only stable, but also allows for mature glycosylation and cellsurface expression of a polypeptide.

Such cell lines of the invention may be cultured using routine methodsto produce a polypeptide of the invention, or may be usedtherapeutically or prophylactically to deliver antibodies of theinvention to a subject. Alternatively, polynucleotides, expressioncassettes or vectors of the invention may be administered to a cell froma subject ex vivo and the cell then returned to the body of the subject.

Pharmaceutical Formulations, Therapeutic Uses and Patient Groups

In another aspect, the present invention provides compositionscomprising molecules of the invention, such as the antibodies,bispecific polypeptides, polynucleotides, vectors and cells describedherein. For example, the invention provides a composition comprising oneor more molecules of the invention, such as one or more antibodiesand/or bispecific polypeptides of the invention, and at least onepharmaceutically acceptable carrier.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Preferably, the carrier is suitable forparenteral, e.g. intravenous, intramuscular or subcutaneousadministration (e.g., by injection or infusion). Depending on the routeof administration, the polypeptide may be coated in a material toprotect the polypeptide from the action of acids and other naturalconditions that may inactivate or denature the polypeptide.

Preferred pharmaceutically acceptable carriers comprise aqueous carriersor diluents. Examples of suitable aqueous carriers that may be employedin the compositions of the invention include water, buffered water andsaline. Examples of other carriers include ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol, and the like), andsuitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials, such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol, or sodium chloride in the composition.

A composition of the invention also may include a pharmaceuticallyacceptable anti-oxidant. These compositions may also contain adjuvantssuch as preservatives, wetting agents, emulsifying agents and dispersingagents. Prevention of presence of microorganisms may be ensured both bysterilization procedures, supra, and by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration.

Sterile injectable solutions can be prepared by incorporating the activeagent (e.g. polypeptide) in the required amount in an appropriatesolvent with one or a combination of ingredients enumerated above, asrequired, followed by sterilization microfiltration. Generally,dispersions are prepared by incorporating the active agent into asterile vehicle that contains a basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum drying and freeze-drying(lyophilization) that yield a powder of the active agent plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Particularly preferred compositions are formulated for systemicadministration or for local administration. Local administration may beat the site of a tumour or into a tumour draining lymph node. Thecomposition may preferably be formulated for sustained release over aperiod of time. Thus the composition may be provided in or as part of amatrix facilitating sustained release. Preferred sustained releasematrices may comprise a montanide or γ-polyglutamic acid (PGA)nanoparticles. Localised release of a polypeptide of the invention,optionally over a sustained period of time, may reduce potentialautoimmune side-effects associated with administration of a CTLA-4antagonist.

Compositions of the invention may comprise additional active ingredientsas well as a polypeptide of the invention. As mentioned above,compositions of the invention may comprise one or more polypeptides ofthe invention. They may also comprise additional therapeutic orprophylactic agents.

Also within the scope of the present invention are kits comprisingpolypeptides or other compositions of the invention and instructions foruse. The kit may further contain one or more additional reagents, suchas an additional therapeutic or prophylactic agent as discussed above.

The polypeptides in accordance with the present invention maybe used intherapy or prophylaxis. In therapeutic applications, polypeptides orcompositions are administered to a subject already suffering from adisorder or condition, in an amount sufficient to cure, alleviate orpartially arrest the condition or one or more of its symptoms. Suchtherapeutic treatment may result in a decrease in severity of diseasesymptoms, or an increase in frequency or duration of symptom-freeperiods. An amount adequate to accomplish this is defined as“therapeutically effective amount”. In prophylactic applications,polypeptides or compositions are administered to a subject not yetexhibiting symptoms of a disorder or condition, in an amount sufficientto prevent or delay the development of symptoms. Such an amount isdefined as a “prophylactically effective amount”. The subject may havebeen identified as being at risk of developing the disease or conditionby any suitable means.

In particular, antibodies and bispecific polypeptides of the inventionmay be useful in the treatment or prevention of cancer. Accordingly, theinvention provides an antibody or bispecific polypeptide of theinvention for use in the treatment or prevention of cancer. Theinvention also provides a method of treating or preventing cancercomprising administering to an individual a polypeptide of theinvention. The invention also provides an antibody or bispecificpolypeptide of the invention for use in the manufacture of a medicamentfor the treatment or prevention of cancer.

The cancer may be prostate cancer, breast cancer, colorectal cancer,pancreatic cancer, ovarian cancer, lung cancer, cervical cancer,rhabdomyosarcoma, neuroblastoma, multiple myeloma, leukemia, acutelymphoblastic leukemia, melanoma, bladder cancer, gastric cancer, headand neck cancer, liver cancer, skin cancer, lymphoma or glioblastoma.

An antibody or bispecific polypeptide of the present invention, or acomposition comprising said antibody or said polypeptide, may beadministered via one or more routes of administration using one or moreof a variety of methods known in the art. As will be appreciated by theskilled artisan, the route and/or mode of administration will varydepending upon the desired results. Systemic administration or localadministration are preferred. Local administration may be at the site ofa tumour or into a tumour draining lymph node. Preferred modes ofadministration for polypeptides or compositions of the invention includeintravenous, intramuscular, intradermal, intraperitoneal, subcutaneous,spinal or other parenteral modes of administration, for example byinjection or infusion. The phrase “parenteral administration” as usedherein means modes of administration other than enteral and topicaladministration, usually by injection. Alternatively, a polypeptide orcomposition of the invention can be administered via a non-parenteralmode, such as a topical, epidermal or mucosal mode of administration.

A suitable dosage of an antibody or polypeptide of the invention may bedetermined by a skilled medical practitioner. Actual dosage levels ofthe active ingredients in the pharmaceutical compositions of the presentinvention may be varied so as to obtain an amount of the activeingredient which is effective to achieve the desired therapeuticresponse for a particular patient, composition, and mode ofadministration, without being toxic to the patient. The selected dosagelevel will depend upon a variety of pharmacokinetic factors includingthe activity of the particular polypeptide employed, the route ofadministration, the time of administration, the rate of excretion of thepolypeptide, the duration of the treatment, other drugs, compoundsand/or materials used in combination with the particular compositionsemployed, the age, sex, weight, condition, general health and priormedical history of the patient being treated, and like factors wellknown in the medical arts.

A suitable dose of an antibody or polypeptide of the invention may be,for example, in the range of from about 0.1 μg/kg to about 100 mg/kgbody weight of the patient to be treated. For example, a suitable dosagemay be from about 1 μg/kg to about 10 mg/kg body weight per day or fromabout 10 g/kg to about 5 mg/kg body weight per day.

Dosage regimens may be adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier.

Antibodies or polypeptides may be administered in a single dose or inmultiple doses. The multiple doses may be administered via the same ordifferent routes and to the same or different locations. Alternatively,antibodies or polypeptides can be administered as a sustained releaseformulation as described above, in which case less frequentadministration is required. Dosage and frequency may vary depending onthe half-life of the polypeptide in the patient and the duration oftreatment that is desired. The dosage and frequency of administrationcan also vary depending on whether the treatment is prophylactic ortherapeutic. In prophylactic applications, a relatively low dosage maybe administered at relatively infrequent intervals over a long period oftime. In therapeutic applications, a relatively high dosage may beadministered, for example until the patient shows partial or completeamelioration of symptoms of disease.

Combined administration of two or more agents may be achieved in anumber of different ways. In one embodiment, the antibody or polypeptideand the other agent may be administered together in a singlecomposition. In another embodiment, the antibody or polypeptide and theother agent may be administered in separate compositions as part of acombined therapy. For example, the modulator may be administered before,after or concurrently with the other agent.

An antibody, polypeptide or composition of the invention may also beused in a method of increasing the activation of a population of cellsexpressing the first and second T cell target, the method comprisingadministering to said population of cells a polypeptide or compositionof the invention under conditions suitable to permit interaction betweensaid cell and a polypeptide of the invention. The population of cellstypically comprises at least some cells which express the first T celltarget, typically T cells, and at least some cells which express thesecond T cell target. The method is typically carried out ex vivo.

For example, an antibody, polypeptide or composition of the inventionmay also be used in a method of increasing the activation of apopulation of cells expressing human OX40 and human CTLA-4, the methodas described above.

Alternatively an antibody, polypeptide or composition of the inventionmay also be used in a method of increasing the activation of apopulation of cells expressing human OX40 and human CD137, the method asdescribed above.

Alternatively an antibody, polypeptide or composition of the inventionmay also be used in a method of increasing the activation of apopulation of cells expressing human CTLA-4 and human CD137, the methodas described above.

Binding Domains for CD137

The bispecific polypeptides of the invention may comprise a bindingdomain which is specific for CD137.

The antibody, or antigen binding fragment thereof, that bindsspecifically to CD137 has certain preferred binding characteristics andfunctional effects, which are explained in more detail below. Saidantibody, or antigen binding fragment thereof, preferably retains thesebinding characteristics and functional effects when incorporated as partof a bispecific antibody of the invention. The invention also providessaid antibody as an antibody or antigen-binding fragment thereof inisolated form, i.e. independently of a bispecific antibody of theinvention.

The anti-CD137 antibody preferably specifically binds to CD137, i.e. itbinds to CD137 but does not bind, or binds at a lower affinity, to othermolecules. The term CD137 as used herein typically refers to humanCD137. The sequence of human CD137 is set out in SEQ ID NO: 148(corresponding to GenBank: AAH06196.1). The antibody may have somebinding affinity for CD137 from other mammals, such as CD137 from anon-human primate, for example Macaca fascicularis (cynomolgus monkey).The antibody preferably does not bind to murine CD137 and/or does notbind to other human TNFR superfamily members, for example human OX40 orCD40.

The antibody has the ability to bind to CD137 in its native state and inparticular to CD137 localised on the surface of a cell. Preferably, theantibody will bind specifically to CD137.

That is, an antibody of the invention will preferably bind to CD137 withgreater binding affinity than that at which it binds to anothermolecule.

“Localised on the surface of a cell” is as defined previously.

The antibody may modulate the activity of a cell expressing CD137,wherein said modulation is an increase or decrease in the activity ofsaid cell. The cell is typically a T cell. The antibody may increase theactivity of a CD4+ or CD8+ effector cell, or may decrease the activityof, or deplete, a regulatory T cell (T reg). In either case, the neteffect of the antibody will be an increase in the activity of effector Tcells, particularly CD4+, CD8+ or NK effector T cells. Methods fordetermining a change in the activity of effector T cells are well knownand are as described earlier.

The antibody preferably causes an increase in activity in a CD8+ T cellin vitro, optionally wherein said increase in activity is an increase inproliferation, IFN-γ production and/or IL-2 production by the T cell.The increase is preferably at least 2-fold, more preferably at least10-fold and even more preferably at least 25-fold higher than the changein activity caused by an isotype control antibody measured in the sameassay.

The antibody preferably binds to human CD137 with a Kd value which isless than 10×10⁻⁹M or less than 7×10⁻⁹M, more preferably less than 4, or2×10⁻⁹M, most preferably less than 1.2×10⁻⁹M.

For example, the antibody preferably does not bind to murine CD137 orany other TNFR superfamily member, such as OX40 or CD40. Therefore,typically, the Kd for the antibody with respect to human CD137 will be2-fold, preferably 5-fold, more preferably 10-fold less than Kd withrespect to the other, non-target molecule, such as murine CD137, otherTNFR superfamily members, or any other unrelated material oraccompanying material in the environment. More preferably, the Kd willbe 50-fold less, even more preferably 100-fold less, and yet morepreferably 200-fold less.

The value of this dissociation constant can be determined directly bywell-known methods, as described earlier. A competitive binding assaycan also be conducted, as described earlier.

An antibody of the invention is preferably capable of binding to itstarget with an affinity that is at least two-fold, 10-fold, 50-fold,100-fold or greater than its affinity for binding to another non-targetmolecule.

In summary therefore, the anti-CD137 antibody preferably exhibits atleast one of the following functional characteristics:

-   -   I. binding to human CD137 with a K_(D) value which is less than        10×10⁻⁹M, more preferably less than 1.2×10⁻⁹M; and    -   II. is capable of causing an increase in activity in a CD8+ T        cell in vitro, optionally wherein said increase in activity is        an increase in proliferation, IFN-γ production and/or IL-2        production by the T cell. The increase is preferably at least        2-fold, more preferably at least 10-fold and even more        preferably at least 25-fold higher than the change in activity        caused by an isotype control antibody measured in the same        assay.

The antibody is specific for CD137, typically human CD137 and maycomprise any one, two, three, four, five or all six of the exemplary CDRsequences of any corresponding pair of rows in Tables I(1) and I(2).

For example, the antibody may comprise any one, two, three, four, fiveor all six of the exemplary CDR sequences of the first rows of TableI(1) and Table I(2) (SEQ ID NOs: 207, 212, 217, 80, 81, 222)

Alternatively the antibody may comprise any one, two, three, four, fiveor all six of the exemplary CDR sequences of the second, third, fourthor fifth rows of Tables I(1) and I(2).

Preferred anti-CD137 antibodies may comprise at least a heavy chain CDR3as defined in any individual row of Table I(1) and/or a light chain CDR3as defined in in any individual row of Table I(2). The antibody maycomprise all three heavy chain CDR sequences shown in an individual rowof Table I(1) (that is, all three heavy chain CDRs of a given “VHnumber”) and/or all three light chain CDR sequences shown in anindividual row of Table I (2) (that is, all three light chain CDRs of agiven “VL number”).

Examples of complete heavy and light chain variable region amino acidsequences of anti-CD137 antibodies are shown in Table H. Exemplarynucleic acid sequences encoding each amino acid sequence are also shown.SEQ ID NOs 177 to 196 refer to the relevant amino acid and nucleotidesequences of anti-CD137 antibodies. The numbering of said VH and VLregions in Table H corresponds to the numbering system used as in TableI(1) and (2). Thus, for example, the amino acid sequence for “1205,light chain VL” is an example of a complete VL region sequencecomprising all three CDRs of VL number 1205 shown in Table I(2) and theamino acid sequence for “1204, heavy chain VH” is an example of acomplete VH region sequence comprising all three CDRs of VH number 1204shown in Table I(1).

Preferred anti-CD137 antibodies of the invention include a VH regionwhich comprises all three CDRs of a particular VH number and a VL regionwhich comprises all three CDRs of a particular VL number. For example:an antibody may comprise all three CDRs of VH number 1204 and all threeCDRs of VL number 1205. Such an antibody may be referred to as1204/1205. Such an antibody may preferably comprise the correspondingcomplete VH and VL sequences of 1204 and 1205 as shown in Table H (SEQID NOs: 179 and 177).

An antibody may alternatively comprise all three CDRs of VH number 1214and all three CDRs of VL number 1215. Such an antibody may be referredto as 1214/1215. Such an antibody may preferably comprise thecorresponding complete VH and VL sequences of 1214 and 1215 as shown inTable H (SEQ ID NOs: 181 and 183).

An antibody may alternatively comprise all three CDRs of VH number 1618and all three CDRs of VL number 1619. Such an antibody may be referredto as 1618/1619. Such an antibody may preferably comprise thecorresponding complete VH and VL sequences of 1618 and 1619 as shown inTable H (SEQ ID NOs: 185 and 187).

An antibody may alternatively comprise all three CDRs of VH number 1620and all three CDRs of VL number 1621. Such an antibody may be referredto as 1620/1621. Such an antibody may preferably comprise thecorresponding complete VH and VL sequences of 1620 and 1621 as shown inTable H (SEQ ID NOs: 189 and 191)

An antibody may alternatively comprise all three CDRs of VH number 1626and all three CDRs of VL number 1627. Such an antibody may be referredto as 1626/1627. Such an antibody may preferably comprise thecorresponding complete VH and VL sequences of 1626 and 1627 as shown inTable H (SEQ ID NOs: 193 and 195)

The anti-CD137 antibody may bind to the same epitope as any of thespecific anti-CD137 antibodies described herein. Preferably it binds tothe same epitope as any one of the antibodies designated 1204/1205,1214/1215, 1618/1619, 1620/1621, and 1626/1627.

Binding Domains for CTLA-4

The bispecific polypeptides of the invention may comprise a bindingdomain specific for CTLA-4.

CD86 and CD80 may be referred to herein as B7 proteins (B7-2 and B7-1respectively). These proteins are expressed on the surface of antigenpresenting cells and interact with the T cell receptors CD28 and CTLA-4.The binding of the B7 molecules to CD28 promotes T cell activation whilebinding of B7 molecules to CTLA-4 switches off the activation of the Tcell. The interaction between the B7 proteins with CD28 and/or CTLA-4constitutes a costimulatory signalling pathway which plays an importantrole in immune activation and regulation. Thus, the B7 molecules arepart of a pathway, amenable to manipulation in order to uncouple immuneinhibition, thereby enhancing immunity in patients.

The CD86 protein is a monomer and consists of two extracellularimmunoglobulin superfamily domains. The receptor binding domain of CD86has a typical IgV-set structure, whereas the membrane proximal domainhas a C1-set like structure. The structures of CD80 and CD86 have beendetermined on their own or in complex with CTLA-4. The contact residueson the CD80 and CD86 molecules are in the soluble extracellular domain,and mostly located in the beta-sheets and not in the (CDR-like) loops.

SEQ ID NO: 3 is the amino acid sequence of the monomeric solubleextracellular domain of human wild-type CD86. This wild type sequencemay optionally lack Alanine and Proline at the N terminus, i.e.positions 24 and 25. These amino acids may be referred to herein as A24and P25 respectively.

A bispecific polypeptide of the invention may incorporate as apolypeptide binding domain a domain which is specific for CTLA-4, a“CTLA-4 binding domain”. Suitable examples of such binding domains aredisclosed in WO 2014/207063, the contents of which are incorporated byreference. The binding domain specific for CTLA-4 may also bind to CD28.The term CTLA-4 as used herein typically refers to human CTLA-4 and theterm CD28 as used herein typically refers to human CD28. The sequencesof human CTLA-4 and human CD28 are set out in SEQ ID NOs: 1 and 2respectively. The CTLA-4 binding domain of the polypeptide of thepresent invention may have some binding affinity for CTLA-4 or CD28 fromother mammals, for example primate or murine CTLA-4 or CD28.

The CTLA-4 binding domain has the ability to bind to CTLA-4 in itsnative state and in particular to CTLA-4 localised on the surface of acell.

“Localised on the surface of a cell” is as defined above.

The CTLA-4 binding domain part of the polypeptide of the invention maycomprise or consist of:

-   -   (i) the amino acid sequence of SEQ ID NO: 3; or    -   (ii) an amino acid sequence in which at least one amino acid is        changed when compared to the amino acid sequence of SEQ ID NO: 3        provided that said binding domain binds to human CTLA-4 with        higher affinity than wild-type human CD86.

In other words, the CTLA-4 binding domain is a polypeptide bindingdomain specific for human CTLA-4 which comprises or consists of (i) themonomeric soluble extracellular domain of human wild-type CD86, or (ii)a polypeptide variant of said soluble extracellular domain, providedthat said polypeptide variant binds to human CTLA-4 with higher affinitythan wild-type human CD86.

Accordingly, the CTLA-4 binding domain of the polypeptide of theinvention may have the same target binding properties as human wild-typeCD86, or may have different target binding properties compared to thetarget binding properties of human wild-type CD86. For the purposes ofcomparing such properties, “human wild-type CD86” typically refers tothe monomeric soluble extracellular domain of human wild-type CD86 asdescribed in the preceding section.

Human wild-type CD86 specifically binds to two targets, CTLA-4 and CD28.Accordingly, the binding properties of the CTLA-4 binding domain of thepolypeptide of the invention may be expressed as an individual measureof the ability of the polypeptide to bind to each of these targets. Forexample, a polypeptide variant of the monomeric extracellular domain ofhuman wild-type CD86 preferably binds to CTLA-4 with a higher bindingaffinity than that of wild-type human CD86 for CTLA-4. Such apolypeptide may optionally also bind to CD28 with a lower bindingaffinity than that of wild-type human CD86 for CD28.

The CTLA-4 binding domain of the polypeptide of the invention is apolypeptide binding domain specific for CTLA-4. This means that it bindsto CTLA-4 preferably with a greater binding affinity than that at whichit binds to another molecule. The CTLA-4 binding domain preferably bindsto CTLA-4 with the same or with a higher affinity than that of wild-typehuman CD86 for CTLA-4.

Preferably, the Kd of the CTLA-4 binding domain of the polypeptide ofthe invention for human CTLA-4 will be at least 2-fold, at least2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least4.5-fold, at least 5-fold, at least 5.5-fold, at least 8-fold or atleast 10-fold less than the Kd of wild-type human CD86 for human CTLA-4.Most preferably, the Kd of the CTLA-4 binding domain for human CTLA-4will be at least 5-fold or at least 10-fold less than the Kd ofwild-type human CD86 for human CTLA-4. A preferred method fordetermining the Kd of a polypeptide for CTLA-4 is SPR analysis, e.g.with a Biacore™ system. Suitable protocols for the SPR analysis ofpolypeptides are set out in the Examples.

Preferably, the EC50 of the CTLA-4 binding domain of the polypeptide ofthe invention for human CTLA-4 will be at least 1.5-fold, at least2-fold, at least 3-fold, at least 5-fold, at least 10-fold, at least12-fold, at least 14-fold, at least 15-fold, at least 17-fold, at least20-fold, at least 25-fold or at least 50-fold less than the EC50 ofwild-type human CD86 for human CTLA-4 under the same conditions. Mostpreferably, the EC50 of the CTLA-4 binding domain for human CTLA-4 willbe at least 10-fold or at least 25-fold less than the EC50 of wild-typehuman CD86 for human CTLA-4 under the same conditions. A preferredmethod for determining the EC50 of a polypeptide for CTLA-4 is viaELISA. Suitable ELISA assays for use in the assessment of the EC50 ofpolypeptides are set out in the Examples.

Preferably, the IC50 of the CTLA-4 binding domain of the polypeptide ofthe invention when competing with wild-type human CD86 for binding tohuman CTLA-4 will be at least 2-fold, at least 3-fold, at least 4-fold,at least 5-fold, at least 10-fold, at least 13-fold, at least 15-fold,at least 50-fold, at least 100-fold, or at least 300-fold less than theIC50 of wild-type human CD86 under the same conditions. Most preferably,the IC50 of the CTLA-4 binding domain will be at least 10-fold or atleast 300-fold less than the IC50 of wild-type human CD86 under the sameconditions. A preferred method for determining the IC50 of a polypeptideof the invention is via ELISA. Suitable ELISA assays for use in theassessment of the IC50 of polypeptides of the invention are set out inthe Examples.

The CTLA-4 binding domain of the polypeptide of the invention may alsobind specifically to CD28. That is, the CTLA-4 binding domain may bindto CD28 with greater binding affinity than that at which it binds toanother molecule, with the exception of CTLA-4. The CTLA-4 bindingdomain may bind to human CD28 with a lower affinity than that ofwild-type human CD86 for human CD28. Preferably, the Kd of the CTLA-4binding domain for human CD28 will be at least 2-fold, preferably atleast 5-fold, more preferably at least 10-fold higher than the Kd ofwild-type human CD86 for human CD28.

The binding properties of the CTLA-4 binding domain of the polypeptideof the invention may also be expressed as a relative measure of theability of a polypeptide to bind to the two targets, CTLA-4 and CD28.That is, the binding properties of the CTLA-4 binding domain may beexpressed as a relative measure of the ability of the polypeptide tobind to CTLA-4 versus its ability to bind to CD28. Preferably the CTLA-4binding domain has an increased relative ability to bind to CTLA-4versus CD28, when compared to the corresponding relative ability ofhuman wild-type CD86 to bind to CTLA-4 versus CD28.

When the binding affinity of a polypeptide for both CTLA-4 and CD28 isassessed using the same parameter (e.g. Kd, EC50), then the relativebinding ability of the polypeptide for each target may be expressed as asimple ratio of the values of the parameter for each target. This ratiomay be referred to as the binding ratio or binding strength ratio of apolypeptide. For many parameters used to assess binding affinity (e.g.Kd, EC50), a lower value indicates a higher affinity. When this is thecase, the ratio of binding affinities for CTLA-4 versus CD28 ispreferably expressed as a single numerical value calculated according tothe following formula:

Binding ratio=[binding affinity for CD28]÷[binding affinity for CTLA-4]

Alternatively, if binding affinity is assessed using a parameter forwhich a higher value indicates a higher affinity, the inverse of theabove formula is preferred. In either context, the CTLA-4 binding domainof the polypeptide of the invention preferably has a higher bindingratio than human wild-type CD86. It will be appreciated that directcomparison of the binding ratio for a given polypeptide to the bindingratio for another polypeptide typically requires that the sameparameters be used to assess the binding affinities and calculate thebinding ratios for both polypeptides.

Preferably, the binding ratio for a polypeptide is calculated bydetermining the Kd of the polypeptide for each target and thencalculating the ratio in accordance with the formula [Kd for CD28]÷[Kdfor CTLA-4]. This ratio may be referred to as the Kd binding ratio of apolypeptide. A preferred method for determining the Kd of a polypeptidefor a target is SPR analysis, e.g. with a Biacore™ system. Suitableprotocols for the SPR analysis of polypeptides of the invention are setout in the Examples. The binding ratio of the CTLA-4 binding domain ofthe polypeptide of the invention calculated according to this method ispreferably at least 2-fold or at least 4-fold higher than the bindingratio of wild-type human CD86 calculated according to the same method.

Alternatively, the binding ratio for a polypeptide may be calculated bydetermining the EC50 of the polypeptide for each target and thencalculating the ratio in accordance with the formula [EC50 forCD28]÷[EC50 for CTLA-4]. This ratio may be referred to as the EC50binding ratio of a polypeptide. A preferred method for determining theEC50 of a polypeptide for a target is via ELISA. Suitable ELISA assaysfor use in the assessment of the EC50 of polypeptides of the inventionare set out in the Examples. The binding ratio of the CTLA-4 bindingdomain of the polypeptide of the invention calculated according to thismethod is at least 2-fold, at least 3-fold, at least 4-fold, at least5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least9-fold or at least 10-fold higher than the binding ratio of wild-typehuman CD86 calculated according to the same method.

The CTLA-4 binding domain of the polypeptide of the invention may havethe ability to cross-compete with another polypeptide for binding toCTLA-4. For example, the CTLA-4 binding domain may cross-compete with apolypeptide having the amino acid sequence of any one of SEQ ID NOs: 6to 24 for binding to CTLA-4. Such cross-competing polypeptides may beidentified in standard binding assays. For example, SPR analysis (e.g.with a Biacore™ system), ELISA assays or flow cytometry may be used todemonstrate cross-competition.

In addition to the above functional characteristics, the CTLA-4 bindingdomain of the polypeptide of the invention has certain preferredstructural characteristics. The CTLA-4 binding domain either comprisesor consists of (i) the monomeric soluble extracellular domain of humanwild-type CD86, or (ii) a polypeptide variant of said solubleextracellular domain, provided that said polypeptide variant binds tohuman CTLA-4 with higher affinity than wild-type human CD86.

A polypeptide variant of the monomeric soluble extracellular domain ofhuman wild-type CD86 comprises or consists of an amino acid sequencewhich is derived from that of human wild-type CD86, specifically theamino acid sequence of the soluble extracellular domain of humanwild-type CD86 (SEQ ID NO: 3), optionally lacking A24 and P25. Inparticular, a variant comprises an amino acid sequence in which at leastone amino acid is changed when compared to the amino acid sequence ofSEQ ID NO: 3 (or said sequence lacking A24 and P25). By “changed” it ismeant that at least one amino acids is deleted, inserted, or substitutedcompared to the amino acid sequence of SEQ ID NO: 3 (or said sequencelacking A24 and P25). By “deleted” it is meant that the at least oneamino acid present in the amino acid sequence of SEQ ID NO: 3 (or saidsequence lacking A24 and P25) is removed, such that the amino acidsequence is shortened by one amino acid. By “inserted” it is meant thatthe at least one additional amino acid is introduced into the amino acidsequence of SEQ ID NO: 3 (or said sequence lacking A24 and P25), suchthat the amino acid sequence is lengthened by one amino acid. By“substituted” it is meant that the at least one amino acid in the aminoacid sequence of SEQ ID NO: 3 (or said sequence lacking A24 and P25) isreplaced with an alternative amino acid.

Typically, at least 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids are changedwhen compared to the amino acid sequence of SEQ ID NO: 3 (or saidsequence lacking A24 and P25). Typically, no more than 10, 9, 8, 7, 6,5, 4, 2 or 1 amino acids are changed when compared to the amino acidsequence of SEQ ID NO: 3 (or said sequence lacking A24 and P25). It willbe appreciated that any of these lower limits may be combined with anyof these upper limits to define a range for the permitted number ofchanges compared to the amino acid sequence of SEQ ID NO: 3 (or saidsequence lacking A24 and P25). Thus, for example, a polypeptide of theinvention may comprise an amino acid sequence in which the permittednumber of amino acid changes compared to the amino acid sequence of SEQID NO: 3 (or said sequence lacking A24 and P25) is in the range 2 to 3,2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 3 to 4, 3 to 5,3 to 6, and so on.

It is particularly preferred that at least 2 amino acids are changedwhen compared to the amino acid sequence of SEQ ID NO: 3 (or saidsequence lacking A24 and P25). Preferably, the permitted number of aminoacid changes compared to the amino acid sequence of SEQ ID NO: 3 (orsaid sequence lacking A24 and P25) is in the range 2 to 9, 2 to 8 or 2to 7.

The numbers and ranges set out above may be achieved with anycombination of deletions, insertions or substitutions compared to theamino acid sequence of SEQ ID NO: 3 (or said sequence lacking A24 andP25). For example, there may be only deletions, only insertions, or onlysubstitutions compared to the amino acid sequence of SEQ ID NO: 3 (orsaid sequence lacking A24 and P25), or any mixture of deletions,insertions or substitutions. Preferably the variant comprises an aminoacid sequence in which all of the changes compared to the amino acidsequence of SEQ ID NO: 3 (or said sequence lacking A24 and P25) aresubstitutions. That is, a sequence in which no amino acids are deletedor inserted compared to the sequence of SEQ ID NO: 3 (or said sequencelacking A24 and P25). In the amino acid sequence of a preferred variant,1, 2, 3, 4, 5, 6, 7, or 8 amino acids are substituted when compared tothe amino acid sequence of SEQ ID NO: 3 (or said sequence lacking A24and P25) and no amino acids are deleted or inserted compared to thesequence of SEQ ID NO: 3 (or said sequence lacking A24 and P25).

Preferably the changes compared to the sequence of SEQ ID NO: 3 (or saidsequence lacking A24 and P25) are in the FG loop region (positions 114to 121) and/or the beta sheet region of SEQ ID NO: 3. The strands of thebeta sheet region have the following positions in SEQ ID NO: 3: A:27-31,B:36-37, C:54-58, C′:64-69, C″:72-74, D:86-88, E:95-97, F:107-113,G:122-133.

Most preferably, the changes compared to the sequence of SEQ ID NO: 3(or said sequence lacking A24 and P25) are in one or more of thepositions selected from 32, 48, 49, 54, 74, 77, 79, 103, 107, 111, 118,120, 121, 122, 125, 127 or 134. All numbering of amino acid positionsherein is based on counting the amino acids in SEQ ID NO: 4 startingfrom the N terminus. Thus, the first position at the N terminus of SEQID NO: 3 is numbered 24 (see schematic diagram in FIG. 4).

Particularly preferred insertions include a single additional amino acidinserted between positions 116 and 117 and/or a single additional aminoacid inserted between positions 118 and 119. The inserted amino acid ispreferably Tyrosine (Y), Serine (S), Glycine (G), Leucine (L) orAspartic Acid (D).

A particularly preferred substitution is at position 122, which isArginine (R). The polypeptide of the invention preferably includes anamino acid sequence in which at least position 122 is substitutedcompared to the amino acid sequence of SEQ ID NO: 3 (or said sequencelacking A24 and P25). The most preferred substitution at position 122 isto replace Arginine (R) with Lysine (K) or Asparagine (N), ranked inorder of preference. This substitution may be referred to as R122K/N.

Other preferred substitutions are at positions 107, 121, and 125, whichare Leucine (L), Isoleucine (I) and Glutamic acid (Q), respectively. Inaddition to the substitution at position 122, the polypeptide of theinvention preferably includes an amino acid sequence in which at leastone of the amino acids at positions 107, 121 and 125 is also substitutedcompared to the amino acid sequence of SEQ ID NO: 3 (or said sequencelacking A24 and P25). The amino acid sequence of the polypeptide of theinvention may also be substituted at one or more of positions 32, 48,49, 54, 64, 74, 77, 79, 103, 111, 118, 120, 127 and 134.

The most preferred substitution at position 107 is to replace Leucine(L) with Isoleucine (I), Phenylalanine (F) or Arginine (R), ranked inorder of preference. This substitution may be referred to as L1071/F/R.Similar notation is used for other substitutions described herein. Themost preferred substitution at position 121 is to replace Isoleucine (I)with Valine (V). This substitution may be referred to as I121V.

The most preferred substitution at position 125 is to replace Glutamine(Q) with Glutamic acid (E). This substitution may be referred to asQ125E.

Other substitutions which may be preferred in the amino acid sequence ofthe polypeptide of the invention include: F321, Q48L, S49T, V54I, V64I,K74I/R, S77A, H79D/S/A, K103E, I111V, T118S, M120L, N127S/D and A134T.

Particularly preferred variants of said soluble extracellular domain ofhuman wild-type CD86 comprise or consist of any one of the amino acidsequences of SEQ ID NOs: 6 to 24, as shown in Table C.

The amino acid sequences shown in SEQ ID NOs: 6 to 14 may optionallyinclude the additional residues AP at the N-terminus. The amino acidsequences shown in SEQ ID NOs: 15 to 24 may optionally lack the residuesAP at the N-terminus. In either case, these residues correspond to A24and P25 of SEQ ID NO: 3.

The CTLA-4 binding domain of the polypeptide of the invention maycomprise or consist of any of the above-described variants of saidsoluble extracellular domain of human wild-type CD86. That is, theCTLA-4 binding domain of the polypeptide of the invention may compriseor consist of the amino acid sequence of any one of SEQ ID NOs: 6 to 24,as shown in Table C.

The binding domain may modulate signalling from CTLA-4, for example whenadministered to a cell expressing CTLA-4, such as a T cell. Preferablythe binding domain reduces, i.e. inhibits or blocks, said signalling andthereby increases the activation of said cell. Changes in CTLA-4signalling and cell activation as a result of administration of a testagent (such as the binding domain) may be determined by any suitablemethod. Suitable methods include assaying for the ability ofmembrane-bound CD86 (e.g. on Raji cells) to bind and signal throughCTLA-4 expressed on the surface of T cells, when in the presence of atest agent or in the presence of a suitable control. An increased levelof T cell IL-2 production or an increase in T cell proliferation in thepresence of the test agent relative to the level of T cell IL-2production and/or T cell proliferation in the presence of the control isindicative of reduced signalling through CTLA-4 and increased cellactivation. A typical assay of this type is disclosed in Example 9 ofUS20080233122.

Binding Domains for OX40

The bispecific binding molecules of the invention may incorporate as abinding domain (for example, as B1) any OX40 binding domain, for examplean anti-OX40 antibody.

The antibody, or antigen binding fragment thereof, that bindsspecifically to OX40 has certain preferred binding characteristics andfunctional effects, which are explained in more detail below. Saidantibody, or antigen binding fragment thereof, preferably retains thesebinding characteristics and functional effects when incorporated as partof a bispecific antibody of the invention. This binding domain may alsobe provided independently of the bispecific molecules of the invention.

The antibody preferably specifically binds to OX40, i.e. it binds toOX40 but does not bind, or binds at a lower affinity, to othermolecules. The term OX40 as used herein typically refers to human OX40.The sequence of human OX40 is set out in SEQ ID NO:51 (corresponding toGenBank: NP_003318.1). The antibody may have some binding affinity forOX40 from other mammals, such as OX40 from a non-human primate (forexample Macaca fascicularis (cynomolgus monkey), Macaca mulatta). Theantibody preferably does not bind to murine OX40 and/or does not bind toother human TNFR superfamily members, for example human CD137 or CD40.

The antibody has the ability to bind to OX40 in its native state and inparticular to OX40 localised on the surface of a cell. Preferably, theantibody will bind specifically to OX40. That is, an antibody of theinvention will preferably bind to OX40 with greater binding affinitythan that at which it binds to another molecule.

“Localised on the surface of a cell” is as defined above.

The antibody may modulate the activity of a cell expressing OX40,wherein said modulation is an increase or decrease in the activity ofsaid cell, as defined above. The cell is typically a T cell. Theantibody may increase the activity of a CD4+ or CD8+ effector cell, ormay decrease the activity of a regulatory T cell (T reg), as describedabove.

In either case, the net effect of the antibody will be an increase inthe activity of effector T cells, particularly CD4+ effector T cells.Methods for determining a change in the activity of effector T cells arewell known and are described above.

The antibody preferably binds to human OX40 with a Kd value which isless than 50×10⁻¹⁰M or less than 25×10⁻¹⁰M, more preferably less than10, 9, 8, 7, or 6×10⁻¹⁰M, most preferably less than 5×10⁻¹⁰M.

For example, the antibody preferably does not bind to murine OX40 or anyother TNFR superfamily member, such as CD137 or CD40. Therefore,typically, the Kd for the antibody with respect to human OX40 will be2-fold, preferably 5-fold, more preferably 10-fold less than Kd withrespect to the other, non-target molecule, such as murine OX40, otherTNFR superfamily members, or any other unrelated material oraccompanying material in the environment. More preferably, the Kd willbe 50-fold less, even more preferably 100-fold less, and yet morepreferably 200-fold less.

The value of this dissociation constant can be determined directly bywell-known methods, as described above.

An antibody of the invention is preferably capable of binding to itstarget with an affinity that is at least two-fold, 10-fold, 50-fold,100-fold or greater than its affinity for binding to another non-targetmolecule.

In summary therefore, the antibody preferably exhibits at least one ofthe following functional characteristics:

-   -   I. binding to human OX40 with a K_(D) value which is less than        10×10⁻¹⁰M;    -   II. does not bind to murine OX40;    -   III. does not bind to other human TNFR superfamily members, for        example human CD137 or CD40.

The antibody is specific for OX40, typically human OX40 and may compriseany one, two, three, four, five or all six of the following:

-   -   (a) a heavy chain CDR1 sequence which is 8 amino acids in length        and comprises the consensus sequence: “G, F, T, F, G/Y/S, G/Y/S,        Y/S, Y/S/A”;    -   (b) a heavy chain CDR2 sequence which is 8 amino acids in length        and comprises the consensus sequence: “I, G/Y/S/T, G/S/Y, S/Y,        G/S/Y, G/S/Y, G/S/Y, T”;    -   (c) a heavy chain CDR3 sequence which is 9 to 17 amino acids in        length and which comprises the consensus sequence of: “A, R,        G/Y/S/H, G/Y/F/V/D, G/Y/P/F, −/H/S, −/N/D/H, −/Y/G, −/Y, −/Y,        −/W/A/V, −/A/Y, −/D/A/Y/G/H/N, Y/S/W/A/T, UM/I/F, D, Y”.        Preferred heavy chain CDR3 sequences within this definition        include a CDR3 sequence of 10 amino acids in length which        comprises the consensus sequence “A, R, Y/H, D, Y, A/Y/G, S/W/A,        M/L, D, Y” or a CDR3 sequence of 11 amino acids in length which        comprises the consensus sequence “A, R, G/Y, V/F/Y, P, H, G/Y/H,        Y, F/I, D, Y”;    -   (d) a light chain CDR1 sequence which consists of the sequence:        “Q, S, I, S, S, Y”;    -   (e) a light chain CDR2 sequence which consists of the sequence:        “A, A, S”;    -   (f) a light chain CDR3 sequence which is 8 to 10 amino acids in        length and comprises the consensus sequence: “Q,Q, S/Y/G,        −/Y/H/G, −/S/Y/G/D, S/Y/G/D, S/Y/G/T, P/L, Y/S/H/L/F, T”. A        preferred example a light chain CDR3 sequence within this        definition consists of the sequence “Q, Q, S, Y, S, T, P, Y, T”

The antibody may comprise at least a heavy chain CDR3 as defined in (c)and/or a light chain CDR3 as defined in (f). The antibody may compriseall three heavy chain CDR sequences of (a), (b) and (c) and/or all threelight chain CDR sequences of (d), (e) and (f).

Exemplary CDR sequences are recited in tables A(1) and A(2), SEQ ID NOs:52 to 88.

Preferred anti-OX40 antibodies may comprise at least a heavy chain CDR3as defined in any individual row of Table A(1) and/or a light chain CDR3as defined in in any individual row of Table A(2). The antibody maycomprise all three heavy chain CDR sequences shown in an individual rowof Table A(1) (that is, all three heavy chain CDRs of a given “VHnumber”) and/or all three light chain CDR sequences shown in anindividual row of Table A(2) (that is, all three light chain CDRs of agiven “VL number”).

Examples of complete heavy and light chain variable region amino acidsequences are shown in Table B. Exemplary nucleic acid sequencesencoding each amino acid sequence are also shown. The numbering of saidVH and VL regions in Table B corresponds to the numbering system used asin Table A(1) and (2). Thus, for example, the amino acid sequence for“1167, light chain VL” is an example of a complete VL region sequencecomprising all three CDRs of VL number 1167 shown in Table A(2) and theamino acid sequence for “1166, heavy chain VH” is an example of acomplete VH region sequence comprising all three CDRs of VH number 1166shown in Table A(1).

Preferred anti-OX40 antibodies of the invention include a VH regionwhich comprises all three CDRs of a particular VH number and a VL regionwhich comprises all three CDRs of a particular VL number. For example:

-   -   an antibody may comprise all three CDRs of VH number 1166 and        all three CDRs of VL number 1167. Such an antibody may be        referred to as 1166/1167. Such an antibody may preferably        comprise the corresponding complete VH and VL sequences of 1166        and 1167 as shown in Table B (SEQ ID NOs: 91 and 89).    -   an antibody may comprise all three CDRs of VH number 1170 and        all three CDRs of VL number 1171. Such an antibody may be        referred to as 1170/1171. Such an antibody may preferably        comprise the corresponding complete VH and VL sequences of 1170        and 1171 as shown in Table B (SEQ ID NOs: 95 and 93).    -   an antibody may comprise all three CDRs of VH number 1164 and        all three CDRs of VL number 1135. Such an antibody may be        referred to as 1164/1135. Such an antibody may preferably        comprise the corresponding complete VH and VL sequences of 1164        and 1135 as shown in Table B (SEQ ID NOs: 99 and 97)    -   an antibody may comprise all three CDRs of VH number 1168 and        all three CDRs of VL number 1135. Such an antibody may be        referred to as 1168/1135. Such an antibody may preferably        comprise the corresponding complete VH and VL sequences of 1168        and 1135 as shown in Table B (SEQ ID NOs: 101 and 97)    -   an antibody may comprise all three CDRs of VH number 1482 and        all three CDRs of VL number 1483. Such an antibody may be        referred to as 1482/1483 Such an antibody may preferably        comprise the corresponding complete VH and VL sequences of 1482        and 1483 as shown in Table B (SEQ ID NOs: 105 and 103).    -   an antibody may comprise all three CDRs of VH number 1490 and        all three CDRs of VL number 1135. Such an antibody may be        referred to as 1490/1135. Such an antibody may preferably        comprise the corresponding complete VH and VL sequences of 1490        and 1135 as shown in Table B (SEQ ID NOs: 107 and 97).    -   an antibody may comprise all three CDRs of VH number 1514 and        all three CDRs of VL number 1515. Such an antibody may be        referred to as 1514/1515. Such an antibody may preferably        comprise the corresponding complete VH and VL sequences of 1514        and 1515 as shown in Table B (SEQ ID NOs: 111 and 109).    -   an antibody may comprise all three CDRs of VH number 1520 and        all three CDRs of VL number 1135. Such an antibody may be        referred to as 1520/1135. Such an antibody may preferably        comprise the corresponding complete VH and VL sequences of 1520        and 1135 as shown in Table B (SEQ ID NOs: 113 and 97).    -   an antibody may comprise all three CDRs of VH number 1524 and        all three CDRs of VL number 1525. Such an antibody may be        referred to as 1524/1525. Such an antibody may preferably        comprise the corresponding complete VH and VL sequences of 1524        and 1525 as shown in Table B (SEQ ID NOs: 117 and 115).    -   an antibody may comprise all three CDRs of VH number 1526 and        all three CDRs of VL number 1527. Such an antibody may be        referred to as 1526/1527. Such an antibody may preferably        comprise the corresponding complete VH and VL sequences of 1526        and 1527 as shown in Table B (SEQ ID NOs: 121 and 119).    -   an antibody may comprise all three CDRs of VH number 1542 and        all three CDRs of VL number 1135. Such an antibody may be        referred to as 1542/1135. Such an antibody may preferably        comprise the corresponding complete VH and VL sequences of 1542        and 1135 as shown in Table B (SEQ ID NOs: 123 and 97).

The antibody may comprise a variant or a fragment of any of the specificamino acid sequences recited in Table B, provided that the antibodybinds to human OX40 and exhibits at least one of functionalcharacteristics I to III. Such a variant or fragment may typicallyretain the CDR sequences of the said sequence of Table B.

A fragment of any one of the heavy or light chain amino acid sequencesshown in Table B may comprise at least 7, at least 8, at least 9, atleast 10, at least 12, at least 15, at least 18, at least 20, least 25,at least 50, at least 60, at least 70, at least 80, at least 90 or atleast 100 consecutive amino acids from the said amino acid sequence.

A variant of any one of the heavy or light chain amino acid sequencesshown in Table B may be a substitution, deletion or addition variant ofsaid sequence, as defined above.

The antibody may bind to the same epitope as any of the specificantibodies described herein. Preferably it binds to the same epitope asany one of the antibodies designated 1166/1167, 1170/1171, 1164/1135,1168/1135, 1482/1483, 1490/1135, 1514/1515, 1520/1135, 1524/1525,1526/1527 and 1542/1135.

Exemplary heavy chain constant region amino acid sequences which may becombined with any VH region sequence disclosed herein (to form acomplete heavy chain), such as the IgG1 heavy chain constant regionsequence, are described above.

Exemplary light chain constant region amino acid sequences which may becombined with any VL region sequence disclosed herein (to form acomplete light chain), such as the kappa chain constant region sequence,are described above.

Embodiment of the Invention: Bispecific Polypeptide Specific for CTLA-4and CD137

In an embodiment of the first aspect of the invention, the bispecificpolypeptide has binding domains which are specific for CD137 and CTLA-4,for example B1 is specific for CD137 and B2 is specific for CTLA-4.

These binding domains are as defined above.

The Bispecific Polypeptide of the Embodiment Part 81—Binding DomainSpecific for CD137

The binding domain specific for CD137 is as defined above.

The Bispecific Polypeptide of the Embodiment Part 82—Binding DomainSpecific for CTLA-4

The binding domain specific for CTLA-4 is as defined above.

The Bispecific Polypeptide of the Embodiment

The bispecific polypeptide of the invention is capable of specificallybinding to both human CD137 and human CTLA-4. By “capable ofspecifically binding to both CD137 and CTLA-4”, it is meant that theanti-CTLA-4 part specifically binds to CTLA-4 and the anti-CD137 partspecifically binds to CD137, in accordance with the definitions providedfor each part above. The bispecific polypeptide may comprise any CD137binding domain as described herein linked to any CTLA-4 binding domainas described herein. Preferably the binding characteristics of thedifferent parts for their respective targets are unchanged orsubstantially unchanged when they are present as part of a bispecificantibody of the invention, when compared to said characteristics for theindividual parts when present as separate entities.

Typically this means that the bispecific molecule will have a Kd forCTLA-4 which is preferably substantially the same as the Kd value forCTLA-4 of the CTLA-4 binding domain when present alone. Alternatively,if the bispecific molecule has a Kd for CTLA-4 which is increasedrelative to the Kd for CTLA-4 of the CTLA-4 binding domain when presentalone, then the increase is by no more than 10 fold, preferably no morethan 9 fold, 8 fold, 7 fold, 6 fold, 5 fold, 4 fold, 3 fold or 2 fold.In addition, the bispecific molecule will independently have a Kd forCD137 which is preferably substantially the same as the Kd value forC137 of the CD137 binding domain when present alone. Alternatively, ifthe bispecific molecule has a Kd for CD137 which is increased relativeto the Kd for CD137 of the anti-CD137 antibody when present alone, thenthe increase is by no more than 10 fold, preferably no more than 9 fold,8 fold, 7 fold, 6 fold, 5 fold, 4 fold, 3 fold or 2 fold. Preferred Kdvalues for the individual binding domains are as described above.

It will be appreciated that any of the fold changes in CTLA-4 bindingmay be independently combined with any of the recited fold changes inCD137 binding to describe the binding characteristics of a givenbispecific molecule.

The binding characteristics for CD137 or CTLA-4 of any bispecificpolypeptide of the invention may be assessed by any suitable assay. Inparticular, the assays set out above for each separate part may also beapplied to a bispecific antibody or a combined assay to assesssimultaneous binding to both targets may be used. Suitable assays forassessing the binding characteristics of bispecific polypeptides of theinvention are also set out in the Examples.

The bispecific polypeptide of the embodiment is capable of modulatingthe activity of cells of the immune system to a greater extent than anindividual agonist of CD137 or CTLA-4 alone, or than a combination ofsuch individual agonists. In particular, administration of thebispecific polypeptide produces a higher level of T cell activity, inparticular effector T cell activity, for example CD4+ effector T cellactivity. The increase in effector T cell activity is also morelocalised than that which results from administration of an individualCD137 or CTLA-4 agonist alone (or a combination thereof), because thebispecific polypeptide exerts the greatest effect only in amicroenvironment in which CTLA-4 and CD137 are both highly expressed.Tumours are such a microenvironment. CD137 is expressed in high levelson CD8 T cells and may thus activate them in particular. CD8 T cells areone of the main effector component of an effective tumor response.

The increase in effector T cell activity may result directly fromstimulation of the effector T cells via activation of the CD137 pathwayor via blockade of the CTLA-4 inhibition pathway, or may resultindirectly from depletion or down-regulation of Tregs, thereby reducingtheir immunosuppressive effect. Depletion/down-regulation of Tregs maybe mediated by antibody dependent cellular phagocytosis (ADCP) orantibody dependent cellular cytotoxicity (ADCC) mechanisms. Overall, theresult will be a very powerful, localised immune activation for theimmediate generation of tumoricidal activity.

The cell surface expression pattern of CTLA-4 and CD137 is partlyoverlapping, thus, the bispecific antibodies of the invention may bindto both targets both in cis and in trans. This may result in stimulationthrough CD137 and CTLA-4 in a FcγR-cross-linking independent manner,either by increasing the level of receptor clustering in cis on the samecell, or by creating an artificial immunological synapse between twocells in trans, which in turn may lead to enhanced receptor clusteringand increased signalling in both cells. Overall, the result will be avery powerful, tumor directed immune activation for the generation oftumoricidal activity.

Measurement of the effect of a bispecific polypeptide of the inventionon cells of the immune system may be achieved with any suitable assay.For example, increased activity of effector T cells may be measured byassays as described above in respect of individual components B1 and B2of the bispecific polypeptide, and include measurement of proliferationor IL-2 production by CD4+ and/or CD8+ T cells in the presence of thebispecific polypeptide relative to a control. An increase ofproliferation or IL-2 production relative to control is indicative ofincreased cell activation. A typical assay of this type is disclosed inExample 9 of US20080233122. Assays for cell proliferation and/or IL-2production are well known and are also exemplified in the Examples. Whenassessed in the same assay, the bispecific molecule will typicallyinduce an increase in the activity of an effector T cell which is atleast 1.5 fold higher or at least 2 fold higher, more preferably 3 foldhigher, most preferably 5 fold higher than the increase in activity ofan effector T cell induced by a combination of monospecific agentsbinding to the same targets.

The bispecific molecule potently activates the immune system when in amicroenvironment in which both CD137 and CTLA-4 are highly expressed.Typically, the bispecific molecule will increase the activity of a CD4+or CD8+ effector cell, or may decrease the activity of a regulatory Tcell (T reg). In either case, the net effect of the antibody will be anincrease in the activity of effector T cells, particularly CD4+ effectorT cells. When assessed in the same assay, the bispecific molecule willtypically induce an increase in the activity of an effector T cell whichis at least 1.5 fold higher or at least 1.7 fold higher, more preferably4.5 fold higher, most preferably 7 fold higher than the increase inactivity of an effector T cell induced by a combination of monospecificagents binding to the same targets.

Methods for determining a change in the activity of effector T cells arewell known and are as described earlier. Assays for cell proliferationand/or IL-2 production are well known and are exemplified in theExamples.

For example, the polypeptide may be capable of specifically binding toboth CTLA-4 and CD137, and B1 may be an antibody, or antigen bindingfragment thereof, specific for CD137; and B2 may be a polypeptidebinding domain specific for CTLA-4, which comprises or consists of:

-   -   i) the amino acid sequence of SEQ ID NO: 3; or    -   ii) an amino acid sequence in which at least one amino acid is        changed when compared to the amino acid sequence of SEQ ID NO: 3        provided that said binding domain binds to human CTLA-4 with        higher affinity than wild-type human CD86.

The CTLA-4 specifically bound by the polypeptide may be primate ormurine, preferably human, CTLA-4, and/or the CD137 specifically bound bythe polypeptide may be primate, preferably human, CD137.

Part B1 of the polypeptide of the invention is an antibody, orantigen-binding fragment thereof, which typically comprises at least oneheavy chain (H) and/or at least one light chain (L). Part B2 of thepolypeptide of the invention may be attached to any part of B1, but maytypically be attached to said at least one heavy chain (H) or at leastone light chain (L), preferably at either the N or the C terminus. PartB2 of the polypeptide of the invention may be so attached eitherdirectly or indirectly via any suitable linking molecule (a linker).

Part B1 preferably comprises at least one heavy chain (H) and at leastone light chain (L) and part B2 is preferably attached to the N or the Cterminus of either said heavy chain (H) or said light chain (L). Anexemplary antibody of B1 consists of two identical heavy chains (H) andtwo identical light chains (L). Such an antibody is typically arrangedas two arms, each of which has one H and one L joined as a heterodimer,and the two arms are joined by disulfide bonds between the H chains.Thus, the antibody is effectively a homodimer formed of two H-Lheterodimers. Part B2 of the polypeptide of the invention may beattached to both H chains or both L chains of such an antibody, or tojust one H chain, or just one L chain.

The polypeptide of the invention may therefore alternatively bedescribed as an anti-CD137 antibody, or an antigen binding fragmentthereof, to which is attached at least one polypeptide binding domainspecific for CTLA-4, which comprises or consists of the monomericsoluble extracellular domain of human wild-type CD86 or a variantthereof.

The binding domains of B1 and B2 may be the only binding domains in thepolypeptide of the invention.

The polypeptide of the invention may comprise a polypeptide arrangedaccording to any one of the following formulae, written in the directionN-C:

-   -   (A) L-(X)n-B2;    -   (B) B2-(X)n-L;    -   (C) B2-(X)n-H;    -   (D) H-(X)n-B2;

wherein H is the heavy chain of an antibody (i.e. of B1), L is the lightchain of an antibody (i.e. of B1), X is a linker and n is 0 or 1. Wherethe linker (X) is a peptide, it typically has the amino acid sequenceSGGGGSGGGGS(SEQ ID NO: 47), SGGGGSGGGGSAP (SEQ ID NO: 48), NFSQP (SEQ IDNO:49), KRTVA (SEQ ID NO: 50) or (SG)m, where m=1 to 7. Schematicrepresentations of formulae (A) to (D) are shown in FIG. 1.

The present invention also provides a polypeptide which consists of apolypeptide arranged according to any of formulae (A) to (D). Saidpolypeptide may be provided as a monomer or may be present as acomponent of a multimeric protein, such as an antibody. Said polypeptidemay be isolated. Examples of amino acid sequences of such polypeptidesare shown in Table H. Exemplary nucleic acid sequences encoding eachamino acid sequence are also shown. Exemplary amino acid and nucleotidesequences are recited in SEQ ID NOs 197-206.

Part B2 may be attached to any part of a polypeptide of the invention,or to a linker, by any suitable means. For example, the various parts ofthe polypeptide may be joined by chemical conjugation, such as with apeptide bond. Thus the polypeptide of the invention may comprise orconsist of a fusion protein comprising B1 (or a component part thereof)and B2, optionally joined by a peptide linker. In such a fusion protein,the CD137-binding domain or domains of B1 and the CTLA-4-binding domainor domains of B2 may be the only binding domains.

Other methods for conjugating molecules to polypeptides are known in theart. For example, carbodiimide conjugation (see Bauminger & Wilchek,1980, Methods Enzymol. 70:151-159; the disclosures of which areincorporated herein by reference) may be used to conjugate a variety ofagents, including doxorubicin, to antibodies or peptides. Thewater-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is particularly useful for conjugating a functionalmoiety to a binding moiety. As a further example, conjugation may beachieved by sodium periodate oxidation followed by reductive alkylationof appropriate reactants, or by glutaraldehyde cross-linking. However,it is recognised that, regardless of which method is selected, adetermination should preferably be made that parts B1 and B2 retain orsubstantially retain their target binding properties when present asparts of the polypeptide of the invention.

The same techniques may be used to link the polypeptide of the invention(directly or indirectly) to another molecule. The other molecule may bea therapeutic agent or a detectable label. Suitable therapeutic agentsinclude a cytotoxic moiety or a drug.

A polypeptide of the invention may be provided in isolated orsubstantially isolated form. By substantially isolated, it is meant thatthere may be substantial, but not total, isolation of the polypeptidefrom any surrounding medium. The polypeptides may be mixed with carriersor diluents which will not interfere with their intended use and stillbe regarded as substantially isolated.

Exemplary polypeptides of the invention may comprise or consist of anyone of the amino acid sequences shown in Table H.

Representative polynucleotides which encode examples of a heavy chain orlight chain amino acid sequence of an antibody may comprise or consistof any one of the nucleotide sequences set out in Table H as SEQ ID NOs178, 180, 182, 184, 186, 188, 190, 192, 194 or 196. Representativepolynucleotides which encode the polypeptides shown in Table H maycomprise or consist of the corresponding nucleotide sequences which arealso shown in Table H (intron sequences are shown in lower case) (Forexample, SEQ ID NOs 197, 199, 201, 203 and 205). Representativepolynucleotides which encode examples of part B2 may comprise or consistof any one of SEQ ID NOS: 25 to 43 as shown in Table E.

Embodiment of the Invention: Bispecific Polypeptide Specific for OX40and CTLA-4

In an alternative embodiment of the first aspect of the invention thepolypeptide has binding domains which are specific for OX40 and CTLA-4,for example B1 is specific for OX40 and B2 is specific for CTLA-4.

These binding domains are as defined above.

The Bispecific Polypeptide of the Embodiment Part 81—Antibody Specificfor OX40

The binding domain specific for OX40 is as defined above.

The Bispecific Polypeptide of the Embodiment Part 82—Binding DomainSpecific for CTLA-4

Part B2 of the polypeptide of the invention is a polypeptide bindingdomain specific for CTLA-4, as described above.

The Bispecific Polypeptide of the Embodiment

The bispecific polypeptide of the embodiment is capable of modulatingthe activity of cells of the immune system to a greater extent than anindividual agonist of OX40 or CTLA-4 alone, or than a combination ofsuch individual agonists. In particular, administration of thebispecific polypeptide produces a higher level of effector T cellactivity, particular CD4+ effector T cell activity. The increase ineffector T cell activity is also more localised than that which resultsfrom administration of an individual OX40 or CTLA-4 agonist alone (or acombination thereof), because the bispecific polypeptide exerts thegreatest effect only in a microenvironment in which CTLA-4 and OX40 areboth highly expressed. Tumours are such a microenvironment. Tumorinfiltrating regulatory T cells (Tregs) express high levels of CTLA-4and OX40, and higher than effector T cells (both CD4 and CD8).

The increase in effector T cell activity may result directly fromstimulation of the effector T cells via activation of the OX40 pathwayor via blockade of the CTLA-4 inhibition pathway, or may resultindirectly from depletion or down-regulation of Tregs, thereby reducingtheir immunosuppressive effect. Depletion/down-regulation of Tregs maybe mediated by antibody dependent cellular phagocytosis (ADCP) orantibody dependent cellular cytotoxicity (ADCC) mechanisms. The highexpression of both CTLA-4 and OX40 on Tregs, compared to effector Tcells, may induce a significantly higher killing of Tregs compared tothe monospecific antibodies. Effector T cells, having lower expressionof CTLA-4 and OX40 will not be depleted by this mechanism. Overall, theresult will be a very powerful, localised immune activation for theimmediate generation of tumoricidal activity.

Measurement of the effect of a bispecific polypeptide of the inventionon cells of the immune system may be achieved with any suitable assay.For example, increased activity of effector T cells may be measured byassays as described above in respect of individual components B1 and B2of the bispecific polypeptide, and include measurement of proliferationor IL-2 production by CD4+ and/or CD8+ T cells in the presence of thebispecific polypeptide relative to a control.

The bispecific polypeptide of the invention is capable of specificallybinding to both human CTLA-4 and human OX40, and comprises B1 and B2 asdefined above.

By “capable of specifically binding to both CTLA-4 and OX40”, it ismeant that part B1 specifically binds to OX40 and part B2 specificallybinds to CTLA-4, in accordance with the definitions provided for eachpart above. Preferably the binding characteristics of parts B1 and B2for their respective targets are unchanged or substantially unchangedwhen they are present as part of a polypeptide of the invention, whencompared to said characteristics for parts B1 and B2 when present asseparate entities.

Typically this means that the bispecific molecule will have a Kd forOX40 which is preferably substantially the same as the Kd value for OX40of B1 when present alone. Alternatively, if the bispecific molecule hasa Kd for OX40 which is increased relative to the Kd for OX40 of B1 whenpresent alone, then the increase is by no more than 10 fold, preferablyno more than 9 fold, 8 fold, 7 fold, 6 fold, 5 fold, 4 fold, 3 fold or 2fold. The bispecific molecule preferably binds to human OX40 with a Kdvalue which is less than 50×10⁻¹⁰M, more preferably less than 25×10⁻¹⁰M,most preferably less than 20×10⁻¹⁰M. In addition, the bispecificmolecule will independently have a Kd for CTLA-4 which is preferablysubstantially the same as the Kd value for CTLA4 of B2 when presentalone. Alternatively, if the bispecific molecule has a Kd for CTLA-4which is increased relative to the Kd for CTLA-4 of B2 when presentalone, then the increase is by no more than 3 fold, preferably no morethan 2 fold. The bispecific molecule preferably binds to human CTLA-4with a Kd value which is less than 60×10⁻⁹M, more preferably less than25×10⁻⁹M, most preferably less than 10×10⁻⁹M.

In other words, the bispecific molecule may have a Kd for OX40 which isless than 50×10⁻¹⁰M, 25×10⁻¹⁰M, or 20×10⁻¹⁰M and independent have a Kdfor CTLA-4 which is less than 60×10⁻⁹M, 25×10⁻⁹M, or 10×10⁻⁹M. It willbe appreciated that any of the Kd values recited for OX40 may beindependently combined with any of the Kd values recited for CTLA-4 todescribe the binding characteristics of a given bispecific molecule.Similarly, any of the recited fold changes in OX40 binding may beindependently combined with any of the recited fold changes in CTLA-4binding to describe the binding characteristics of a given bispecificmolecule.

The binding characteristics of parts B1 and B2 when present as part apolypeptide of the invention may be assessed by any suitable assay. Inparticular, the assays set out above for each separate part may also beapplied to B1 and B2 when they are present as part of a polypeptide ofthe invention. Suitable assays for assessing the binding characteristicsof bispecific polypeptides of the invention are also set out in theExamples.

The bispecific molecule potently activates the immune system when in amicroenvironment in which both OX40 and CTLA-4 are highly expressed.Typically, the bispecific molecule will increase the activity of a CD4+or CD8+ effector cell, or may decrease the activity of a regulatory Tcell (T reg). In either case, the net effect of the antibody will be anincrease in the activity of effector T cells, particularly CD4+ effectorT cells. When assessed in the same assay, the bispecific molecule willtypically induce an increase in the activity of an effector T cell whichis at least 1.5 fold higher or at least 1.7 fold higher, more preferably4.5 fold higher, most preferably 7 fold higher than the increase inactivity of an effector T cell induced by a combination of monospecificagents binding to the same targets.

Methods for determining a change in the activity of effector T cells arewell known and are as described earlier. Assays for cell proliferationand/or IL-2 production are well known and are exemplified in theExamples.

For example, the polypeptide may be capable of specifically binding toboth CTLA-4 and OX40, and B1 may be an antibody, or antigen bindingfragment thereof, specific for OX40; and B2 may be a polypeptide bindingdomain specific for CTLA-4, which comprises or consists of:

-   -   i) the amino acid sequence of SEQ ID NO: 3; or    -   ii) an amino acid sequence in which at least one amino acid is        changed when compared to the amino acid sequence of SEQ ID NO: 3        provided that said binding domain binds to human CTLA-4 with        higher affinity than wild-type human CD86.

The CTLA-4 specifically bound by the polypeptide may be primate ormurine, preferably human, CTLA-4, and/or the OX40 specifically bound bythe polypeptide may be primate, preferably human, OX40.

Part B1 of the polypeptide of the invention is an antibody, orantigen-binding fragment thereof, which typically comprises at least oneheavy chain (H) and/or at least one light chain (L). Part B2 of thepolypeptide of the invention may be attached to any part of B1, but maytypically be attached to said at least one heavy chain (H) or at leastone light chain (L), preferably at either the N or the C terminus. PartB2 of the polypeptide of the invention may be so attached eitherdirectly or indirectly via any suitable linking molecule (a linker).

Part B1 preferably comprises at least one heavy chain (H) and at leastone light chain (L) and part B2 is preferably attached to the N or the Cterminus of either said heavy chain (H) or said light chain (L). Anexemplary antibody of B1 consists of two identical heavy chains (H) andtwo identical light chains (L). Such an antibody is typically arrangedas two arms, each of which has one H and one L joined as a heterodimer,and the two arms are joined by disulfide bonds between the H chains.Thus, the antibody is effectively a homodimer formed of two H-Lheterodimers. Part B2 of the polypeptide of the invention may beattached to both H chains or both L chains of such an antibody, or tojust one H chain, or just one L chain.

The polypeptide of the invention may therefore alternatively bedescribed as an anti-OX40 antibody, or an antigen binding fragmentthereof, to which is attached at least one polypeptide binding domainspecific for CTLA-4, which comprises or consists of the monomericsoluble extracellular domain of human wild-type CD86 or a variantthereof. The binding domains of B1 and B2 may be the only bindingdomains in the polypeptide of the invention.

The polypeptide of the invention may comprise a polypeptide arrangedaccording to any one of the following formulae, written in the directionN-C:

-   -   (A) L-(X)n-B2;    -   (B) B2-(X)n-L;    -   (C) B2-(X)n-H; and    -   (D) H-(X)n-B2;

wherein H is the heavy chain of an antibody (i.e. of B1), L is the lightchain of an antibody (i.e. of B1), X is a linker and n is 0 or 1. Wherethe linker (X) is a peptide, it typically has the amino acid sequenceSGGGGSGGGGS (SEQ ID NO: 47), SGGGGSGGGGSAP (SEQ ID NO: 48), NFSQP (SEQID NO:49), KRTVA (SEQ ID NO: 50) or (SG)m, where m=1 to 7. Schematicrepresentations of formulae (A) to (D) are shown in FIG. 1.

The present invention also provides a polypeptide which consists of apolypeptide arranged according to any of formulae (A) to (D). Saidpolypeptide may be provided as a monomer or may be present as acomponent of a multimeric protein, such as an antibody. Said polypeptidemay be isolated. Examples of amino acid sequences of such polypeptidesare shown in Table D. Exemplary nucleic acid sequences encoding eachamino acid sequence are also shown.

Part B2 may be attached to any part of a polypeptide of the invention,or to a linker, by any suitable means. For example, the various parts ofthe polypeptide may be joined by chemical conjugation, such as with apeptide bond. Thus the polypeptide of the invention may comprise orconsist of a fusion protein comprising B1 (or a component part thereof)and B2, optionally joined by a peptide linker. In such a fusion protein,the OX40-binding domain or domains of B1 and the CTLA-4-binding domainor domains of B2 may be the only binding domains.

Other methods for conjugating molecules to polypeptides are known in theart. For example, carbodiimide conjugation (see Bauminger & Wilchek,1980, Methods Enzymol. 70:151-159; the disclosures of which areincorporated herein by reference) may be used to conjugate a variety ofagents, including doxorubicin, to antibodies or peptides. Thewater-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is particularly useful for conjugating a functionalmoiety to a binding moiety. As a further example, conjugation may beachieved by sodium periodate oxidation followed by reductive alkylationof appropriate reactants, or by glutaraldehyde cross-linking. However,it is recognised that, regardless of which method is selected, adetermination should preferably be made that parts B1 and B2 retain orsubstantially retain their target binding properties when present asparts of the polypeptide of the invention.

The same techniques may be used to link the polypeptide of the invention(directly or indirectly) to another molecule. The other molecule may bea therapeutic agent or a detectable label. Suitable therapeutic agentsinclude a cytotoxic moiety or a drug.

A polypeptide of the invention may be provided in isolated orsubstantially isolated form. By substantially isolated, it is meant thatthere may be substantial, but not total, isolation of the polypeptidefrom any surrounding medium. The polypeptides may be mixed with carriersor diluents which will not interfere with their intended use and stillbe regarded as substantially isolated.

Exemplary polypeptides of the invention may comprise or consist of anyone of the amino acid sequences shown in Table D. In one embodiment thepolypeptide comprises or consists of the amino acid sequence selected offrom within the group SEQ ID NOs 125 to 134, optionally wherein saidpolypeptide is a provided as a component part of an antibody.

Representative polynucleotides which encode examples of a heavy chain orlight chain amino acid sequence of an antibody may comprise or consistof any one of the nucleotide sequences set out in Table B.Representative polynucleotides which encode the polypeptides shown inTable D may comprise or consist of the corresponding nucleotidesequences which are also shown in Table D (intron sequences are shown inlower case). Representative polynucleotides which encode examples ofpart B2 may comprise or consist of any one of SEQ ID NOS: 25 to 43 asshown in Table E.

Embodiment of the Invention: Bispecific Polypeptide for OX40 and CD137

In an alternative embodiment of the first aspect of the invention thepolypeptide has binding domains which are specific for OX40 and CD137.

These binding domains are as described above.

The Bispecific Polypeptide of the Embodiment—Binding Domain Specific forOX40

The binding domain specific for OX40 is as defined above.

The Bispecific Polypeptide of the Embodiment—Binding Domain Specific forCD137

The binding domain specific for CD137 is as defined above, with thepreviously described functional and structural characteristics.

Preferably the antibody is 1204/1205, as previously defined, withreference to the sequences of Tables H and I. The anti-CD137 antibodymay bind to the same epitope as any of the specific anti-CD137antibodies described herein. Preferably it binds to the same epitope asthe antibody designated 1204/1205.

The Bispecific Polypeptide of the Embodiment

The bispecific antibody of the invention is capable of specificallybinding to both human CD137 and human OX40. By “capable of specificallybinding to both CD137 and OX40”, it is meant that the anti-OX40 partspecifically binds to OX40 and the anti-CD137 part specifically binds toCD137, in accordance with the definitions provided for each part above.The bispecific antibody may comprise any anti-CD137 antibody asdescribed herein linked to any anti-OX40 antibody as described herein.Preferably the binding characteristics of the different parts for theirrespective targets are unchanged or substantially unchanged when theyare present as part of a bispecific antibody of the invention, whencompared to said characteristics for the individual parts when presentas separate entities.

Typically this means that the bispecific molecule will have a Kd forOX40 which is preferably substantially the same as the Kd value for OX40of the anti-OX40 antibody when present alone. Alternatively, if thebispecific molecule has a Kd for OX40 which is increased relative to theKd for OX40 of the anti-OX40 antibody when present alone, then theincrease is by no more than 10 fold, preferably no more than 9 fold, 8fold, 7 fold, 6 fold, 5 fold, 4 fold, 3 fold or 2 fold. In addition, thebispecific molecule will independently have a Kd for CD137 which ispreferably substantially the same as the Kd value for C137 of theanti-CD137 antibody when present alone. Alternatively, if the bispecificmolecule has a Kd for CD137 which is increased relative to the Kd forCD137 of the anti-CD137 antibody when present alone, then the increaseis by no more than 10 fold, preferably no more than 9 fold, 8 fold, 7fold, 6 fold, 5 fold, 4 fold, 3 fold or 2 fold.

It will be appreciated that any of the fold changes in OX40 binding maybe independently combined with any of the recited fold changes in CD137binding to describe the binding characteristics of a given bispecificmolecule.

The binding characteristics for CD137 or OX40 of any bispecific antibodyof the invention may be assessed by any suitable assay. In particular,the assays set out above for each separate part may also be applied to abispecific antibody or a combined assay to assess simultaneous bindingto both targets may be used. Suitable assays for assessing the bindingcharacteristics of bispecific polypeptides of the invention are also setout in the Examples.

The bispecific antibody of the invention is capable of modulating theactivity of cells of the immune system to a greater extent than anindividual agonist of OX40 or CD137 alone, or than a combination of suchindividual agonists. In particular, administration of the bispecificantibody produces a higher level of T cell activity. The increase ineffector T cell activity is also more localised than that which resultsfrom administration of an individual OX40 or CD137 agonist alone (or acombination thereof), because the bispecific polypeptide exerts thegreatest effect only in a microenvironment in which CD137 and OX40 areboth highly expressed. Tumours are such a microenvironment.

The cell surface expression pattern of OX40 and CD137 is partlyoverlapping, thus, the bispecific antibodies of the invention may bindto both targets both in cis and in trans. This may result in stimulationthrough CD137 and OX40 in a FcγR-cross-linking independent manner,either by increasing the level of receptor clustering in cis on the samecell, or by creating an artificial immunological synapse between twocells in trans, which in turn may lead to enhanced receptor clusteringand increased signalling in both cells. Overall, the result will be avery powerful, tumor directed immune activation for the generation oftumoricidal activity.

Measurement of the effect of a bispecific antibody of the invention oncells of the immune system may be achieved with any suitable assay. Forexample, increased activity of effector T cells may be measured byassays as described above in respect of the monospecific components ofthe bispecific antibody, and include measurement of proliferation orIL-2 production by CD4+ and/or CD8+ T cells in the presence of thebispecific antibody relative to a control. An increase of proliferationor IL-2 production relative to control is indicative of increased cellactivation. A typical assay of this type is disclosed in Example 9 ofUS20080233122. Assays for cell proliferation and/or IL-2 production arewell known and are also exemplified in the Examples. When assessed inthe same assay, the bispecific molecule will typically induce anincrease in the activity of an effector T cell which is at least 1.5fold higher or at least 2 fold higher, more preferably 3 fold higher,most preferably 5 fold higher than the increase in activity of aneffector T cell induced by a combination of monospecific agents bindingto the same targets.

The invention provides a bispecific antibody comprising an antibodywhich specifically binds to OX40 (an anti-OX40 antibody) and an antibodywhich specifically binds to CD137 (an anti-CD137 antibody), joined toeach other directly or indirectly. By “joined indirectly” it is meantthat another moiety (a linker) links the anti-OX40 antibody to theanti-CD137 antibody. Exemplary linkers include a peptide of amino acidsequence as shown in any one of SEQ ID NOs. 47 to 50, or 144. Thepeptide of sequence GGGGSGGGGSGGGGS (SEQ ID NO: 144) is particularlypreferred.

The anti-OX40 antibody may be attached to any part of the anti-CD137antibody, or to a linker, by any suitable means. For example, thevarious parts of the polypeptide may be joined by chemical conjugation,such as with a peptide bond. Thus the polypeptide of the invention maycomprise or consist of a fusion protein comprising an anti-OX40 antibody(or antigen binding fragment thereof) and an anti-CD137 antibody (orantigen binding fragment thereof), optionally joined by a peptidelinker. In such a fusion protein, the CD137-binding domain or domainsand the OX40-binding domain or domains may be the only binding domainspresent.

Other methods for conjugating molecules to polypeptides are known in theart. For example, carbodiimide conjugation (see Bauminger & Wilchek,1980, Methods Enzymol. 70:151-159; the disclosures of which areincorporated herein by reference) may be used to conjugate a variety ofagents, including doxorubicin, to antibodies or peptides. Thewater-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is particularly useful for conjugating a functionalmoiety to a binding moiety. As a further example, conjugation may beachieved by sodium periodate oxidation followed by reductive alkylationof appropriate reactants, or by glutaraldehyde cross-linking. However,it is recognised that, regardless of which method is selected, adetermination should preferably be made that the anti-OX40 andanti-CD137 parts of the bispecific antibody retain or substantiallyretain their target binding properties when present as parts of thebispecific antibody of the invention.

The same techniques may be used to link any antibody of the invention(directly or indirectly) to another molecule. The other molecule may bea therapeutic agent or a detectable label. Suitable therapeutic agentsinclude a cytotoxic moiety or a drug.

The bispecific antibody of the invention may comprise the anti-OX40antibody (or antigen binding fragment thereof) and the anti-CD137antibody (or antigen binding fragment thereof) arranged together in anysuitable format. It will be appreciated that in any given bispecificformat, the anti-OX40 antibody and the anti-CD137 antibody may eachindependently be a whole antibody or an antigen binding portion thereof.Irrespective of the particular bispecific format used, bispecificantibodies described herein may typically be referred to by a numberingscheme based on the composition of the OX40 binding domain (which may bereferred to as binding domain 1) and the composition of the CD137binding domain (which may be referred to as binding domain 2). Thenumbering scheme is therefore typically in the form VH1/VL1 for the OX40binding domain (binding domain 1) and VH2/VL2 for the CD137 bindingdomain (binding domain 2), written together as VH1/VL1-VH2/VL2. Thus,for example, a bispecific antibody referred to as 1164/1135-1204/1205comprises at least one OX40 binding domain which consists of the VHsequence 1164 and the VL sequence 1135 (i.e. 1164/1135=VH1/VL1), and atleast one CD137 binding domain which consists of the VH sequence 1204and the VL sequence 1205 (i.e. 1204/1205=VH2/VL2). It will beappreciated that this numbering scheme does not reflect the total numberof binding domains present in the bispecific antibody nor the presenceor absence of any constant regions in the bispecific antibody, both ofwhich are determined by the particular format of bispecific antibodythat is used. The total number of binding domains and the presence orabsence of constant regions may be in accordance with any suitablebispecific antibody format known in the art.

Many suitable formats of bispecific antibody are known in the art andthe bispecific antibody of the invention may be in any of these formats.Suitable formats include those described in FIGS. 1 and 19 (see alsoKontermann & Brinkmann, 2015, Drug Discov Today. 838-847; thedisclosures of which are incorporated herein by reference).

In FIG. 19, constant regions are shown as filled light grey; variableheavy chain region VH1 is shown as checkered black and white; variablelight chain region VL1 is shown as filled white; variable heavy chainregion VH2 is shown as filled black; and variable light chain region VL2is shown as white with diagonal lines. Thus, OX40 binding domains(referred to as binding domain 1) are typically represented as a pair ofa checkered black and white domain with a filled white domain; CD137binding domains (referred to as binding domain 2) are typicallyrepresented as a pair of a filled black domain and a white domain withdiagonal lines. However, in all of the formats shown, it will beappreciated that binding domains 1 and 2 may be switched. That is, anOX40 binding domain may occur in any position shown in FIG. 19 for aCD137 domain, and vice versa.

A preferred format for the bispecific antibody is a kih or“knob-in-hole” arrangement, which is the first shown in the second rowof FIG. 19. In this arrangement, the CH3 domain of the heavy chain ofeach antibody is mutated to allow heterodimerisation between a heavychain from the anti-OX40 antibody and a heavy chain from the anti-CD137antibody. Each heavy chain associates with its corresponding light chainto form one complete OX40 binding domain and one complete CD137 bindingdomain. Modifications may be made to the heavy chain CH1 regions topromote association with the correct light chain. Kih format bispecificantibodies are well-known in the art. See for example Ridgway et al1996; Protein Eng 9:617-621, the disclosures of which are incorporatedherein by reference.

Another preferred format for the bispecific antibody of the invention isscFv₂-Fc format, which is the second shown in the second row of FIG. 19.In this arrangement, one scFv specific for each target is fused toconstant immunoglobulin domains. The single chains may be fused to theFc region of the heavy chain, with one specificity fused to theN-terminal end and the other specificity fused to the C-terminal end ofthe Fc region (Park et al., 2000, Mol Immunol 37(18):1123-30; thedisclosures of which are incorporated herein by reference).

Another preferred format for the bispecific antibody of the invention isthe BITE/scFv₂ format which is the third format shown in the second rowof FIG. 19. In this arrangement two scFv, one specific for OX40 and theother specific for CD137, are fused together with a linker (Brischweinet al., 2007, J Immunother 30(8):798-807; the disclosures of which areincorporated herein by reference). The linker may optionally include aprotein that increases solubility and serum half-life, such as humanserum albumin (HSA), creating scFv-HSA-scFv bispecific antibodies, asshown in the fourth row of FIG. 19.

Another preferred format for the bispecific antibody of the invention isdouble variable domain (DVD) immunoglobulins, which is the fourth formatshown in the second row of FIG. 19. In this arrangement the secondvariable domain (VL2) is fused to the first variable light chain (VL1),and the second variable heavy chain (VH2) is fused to the first variableheavy chain (VH1) of an IgG molecule. VH1 and VL1 form binding site 1and VH2 and VL2 form binding site 2, thus creating a bispecific antibody(Wu, 2007, Nat Biotechnol 25(11):1290-7; the disclosures of which areincorporated herein by reference).

Another preferred format for the bispecific antibody of the invention isthe Dual affinity retargeting (DART) format in which the VH1 is fused toVL2 and VH2 fused to VL1 with a short peptide linker forcing them toform VH1/VL1 and VH2/VL2 binding sites. This construct may be stabilizedby formation of a disulphide bridge between the binding sites. The DARTformat may be fused to IgG Fc domains, creating monovalent bispecificantibodies (DART-Fc) or bivalent bispecific antibodies (DART₂-Fc) (Mooreet al., 2011, Blood 117(17):4542-51). The DART, DART-Fc and DART₂-Fcformats are shown in the third row of FIG. 19.

Another preferred format for the bispecific antibody of the invention isbispecific antibodies generated by the dock and lock technology (DNL).cAMP dependent protein kinase A and A kinase anchoring protein can befused to antibodies, Fab fragments or scFv for each target, therebygenerating multivalent bispecific antibodies, e.g. DNL-Fab₃ (Chang etal., 2007, Clin Cancer Res 13(18 Pt 2):5586s-5591s, as shown in thefourth row of FIG. 19; the disclosures of which are incorporated hereinby reference).

A particularly preferred format for the bispecific antibody of theinvention is the scFv-IgG format. Four different possible arrangementsof this format are shown in the top row of FIG. 19. As shown in FIG. 19,in scFv-IgG format the anti-OX40 antibody is a whole IgG molecule andthe anti-CD137 antibody is an scFv antibody connected to the anti-OX40antibody at any one of four general locations (heavy chain constantregion; light chain constant region; heavy chain variable region; lightchain variable region). In each case, the reverse arrangement is alsoenvisaged. That is, with the anti-CD137 antibody as a whole IgG and theanti-OX40 antibody as an scFv connected to the anti-CD137 antibody atany one of the four general locations. In the scFv-IgG format, the wholeIgG molecule may be joined directly to the scFv or may be joinedindirectly via a linker. Exemplary linkers include a peptide of aminoacid sequence as shown in any one of SEQ ID NOs. 47 to 50, or 144, withthe peptide of sequence GGGGSGGGGSGGGGS (SEQ ID NO: 144) particularlypreferred.

In the first scFv-IgG arrangement shown in FIG. 19 (top left of theFigure) the bispecific antibodies comprise two copies of a polypeptidechain which comprises the heavy chain variable sequence VH1 (checkeredblack and white), linked to a heavy chain constant sequence (Hc; filledgrey), linked (optionally via a linker) to an scFv sequence consistingof the heavy chain variable sequence VH2 (filled black) and the lightchain variable sequence VL2 (white with diagonal lines). This chain maybe referred to as VH1-Hc-VH2/VL2 (ordered N terminus-C terminus). Thebispecific antibody also comprises two copies of a smaller chain whichcomprises the light chain variable sequence VL1 (filled white) linked toa light chain constant sequence (Lc; filled grey), which may be referredto as VL1-Lc (ordered N terminus-C terminus). The alternative scFv-IgGarrangements shown in FIG. 19 also comprise two copies each of twodifferent chains, which may be described in similar fashion. Thus,reading from left to right in the top row of FIG. 22, the secondarrangement comprises two VH1-Hc chains and two VL1-Lc-VH2/VL2 chains.The third arrangement comprises two VH1/VH2-VH1-Hc chains and two VL1-Lcchains. The fourth arrangement comprises two VH1-Hc and twoVH1/VH2-VL1-Lc chains.

The most preferred scFv-IgG format for bispecific antibodies of theinvention is the first scFv-IgG arrangement shown in FIG. 19 (top leftof the Figure). Exemplary amino acid sequences for polypeptide chains ofthe type VH1-Hc-VH2/VL2 from this scFv-IgG arrangement are shown inTable F and are used in the bispecific molecules of the Examples. Ineach case, for the amino acid sequences the underlined/bold sequence isVH1, the italic sequence is the heavy chain constant sequence Hc(typically an IgG1 constant domain in the sequences shown, but this maybe replaced with any suitable IgG constant domain such as thosedisclosed herein), the underlined sequence is an optional linker, thebold sequence is VH2 and the bold italic sequence is VL2. Correspondingexemplary nucleotide sequences encoding the polypeptide chains are alsoshown in Table F. The nucleotide sequences include introns. Exemplaryamino acid sequences for polypeptide chains of the type VL1-Lc from thisscFv-IgG arrangement are shown in Table G and are used in the bispecificmolecules of the Examples. In each case, for the amino acid sequencesthe underlined/bold sequence is VL1 and the italic sequence is the lightchain constant sequence Lc (typically kappa, but may be replaced withany suitable light chain constant region). Corresponding exemplarynucleotide sequences encoding the polypeptide chains are also shown inTable G.

The present invention provides a polypeptide comprising or consisting ofany of the amino acid sequences set out in Tables F and G, either aloneor, preferably as part of a monospecific or bispecific antibody. In allof the sequences shown in Tables F and G, the sequences corresponding toheavy or light chain constant regions are exemplary and may be replacedwith any other suitable heavy or light chain constant region sequence.Preferred heavy and light chain constant region sequences are those ofSEQ ID NOs: 135, 136, 137, 138 and 139.

A bispecific or monospecific antibody of the invention may be providedin isolated or substantially isolated form. By substantially isolated,it is meant that there may be substantial, but not total, isolation ofthe polypeptide from any surrounding medium. The polypeptides may bemixed with carriers or diluents which will not interfere with theirintended use and still be regarded as substantially isolated.

An antibody of the invention may be produced by any suitable means. Forexample, all or part of the antibody may be expressed as a fusionprotein by a cell comprising a nucleotide which encodes saidpolypeptide.

Alternatively the individual parts may be produced separately and thensubsequently joined together. Joining may be achieved by any suitablemeans, for example using the chemical conjugation methods and linkersoutlined above. Separate production of individual parts may be achievedby any suitable means. For example by expression from separatenucleotides optionally in separate cells, as is explained in more detailbelow.

Representative polynucleotides which encode all or part of an antibodyof the invention may comprise or consist of any one of the nucleotidesequences set out in Tables B, F, G and H. A suitable polynucleotide mayalternatively be a variant of any of these sequences, as defined above.

FURTHER ASPECTS OF THE INVENTION

A second aspect of the invention comprises a bispecific polypeptideaccording to the first aspect of the invention for use in a method fortreating or preventing a disease or condition in an individual, asdescribed above.

A third aspect of the invention is a method of treating or preventing adisease or condition in an individual, the method comprisingadministering to an individual a bispecific polypeptide according to thefirst or second aspects of the invention, as described above.

One embodiment of the invention is a bispecific polypeptide according tothe second aspect of the invention or a method according to third aspectof the invention wherein the disease or condition is cancer andoptionally wherein the individual is human.

In a further embodiment, the method comprises administering thebispecific antibody systemically or locally, such as at the site of atumour or into a tumour draining lymph node, as described above.

The cancer may be prostate cancer, breast cancer, colorectal cancer,pancreatic cancer, ovarian cancer, lung cancer, cervical cancer,rhabdomyosarcoma, neuroblastoma, multiple myeloma, leukemia, acutelymphoblastic leukemia, melanoma, bladder cancer, gastric cancer, headand neck cancer, liver cancer, skin cancer, lymphoma or glioblastoma.

A fourth aspect of the invention is a polynucleotide encoding at leastone polypeptide chain of a bispecific polypeptide according to the firstor second aspects of the invention, as described above.

A fifth aspect of the invention is a composition comprising a bispecificpolypeptide according to the first or second aspects of the inventionand at least one pharmaceutically acceptable diluent or carrier.

In one embodiment of the invention a polypeptide according to either thefirst or second aspect of the embodiment is conjugated to an additionaltherapeutic moiety.

A sixth aspect of the invention is an antibody specific for CD137 whichis as defined earlier.

EMBODIMENTS OF THE INVENTION

Embodiments of the invention are described in the following paragraphs:

1. A polypeptide capable of specifically binding to both CTLA-4 andOX40, said polypeptide comprising B1 and B2, wherein:

B1 is an antibody, or antigen binding fragment thereof, specific forOX40; and

B2 is a polypeptide binding domain specific for CTLA-4, which comprisesor consists of:

-   -   i) the amino acid sequence of SEQ ID NO: 3; or    -   ii) an amino acid sequence in which at least one amino acid is        changed when compared to the amino acid sequence of SEQ ID NO: 3        provided that said binding domain binds to human CTLA-4 with        higher affinity than wild-type human CD86.

2. A polypeptide according to paragraph 1 in which the CTLA-4specifically bound by the polypeptide is primate or murine, preferablyhuman, CTLA-4, and/or wherein the OX40 specifically bound by thepolypeptide is primate, preferably human, OX40.

3. A polypeptide according to paragraph 1 or 2 in which B1 comprises atleast one heavy chain (H) and/or at least one light chain (L) and B2 isattached to said at least one heavy chain (H) or least one light chain(L).

4. A polypeptide according to paragraph 3 in which B1 comprises:

-   -   at least one heavy chain (H) and at least one light chain (L)        and B2 is attached to either the heavy chain or the light chain;        or    -   two identical heavy chains (H) and two identical light        chains (L) and B2 is attached to both heavy chains or to both        light chains.

5. A polypeptide according to any one of the preceding paragraphs whichcomprises or consists of a polypeptide chain arranged according to anyone of the following formulae, written in the direction N-C:

-   -   (A) L-(X)n-B2;    -   (B) B2-(X)n-L;    -   (C) B2-(X)n-H;    -   (D) H-(X)n-B2;

wherein X is a linker and n is 0 or 1.

6. A polypeptide according to paragraph 5, wherein X is a peptide withthe amino acid sequence SGGGGSGGGGS (SEQ ID NO: 47), SGGGGSGGGGSAP (SEQID NO: 48), NFSQP (SEQ ID NO:49), KRTVA (SEQ ID NO: 50) or (SG)m, wherem=1 to 7.

7. A polypeptide according to any one of the preceding paragraphs, whichbinds to h., human OX40 with a Kd of less than 50×10⁻¹⁰M, 25×10⁻¹⁰M, or20×10⁻¹⁰M and/or which binds to human CTLA-4 with a Kd value which isless than 60×10⁻⁹M, 25×10⁻⁹M, or 10×10⁻⁹M.

8. A polypeptide according to any one of the preceding paragraphs, whichinduces an increase in the activity of an effector T cell, preferably aCD4+ effector T cell, optionally wherein said increase is at least 1.5fold, 4.5 fold or 7 fold higher than the increase in activity of aneffector T cell induced by a combination of B1 and B2 administered tothe T cell as separate molecules.

9. A polypeptide according to paragraph 8, wherein said increase in Tcell activity is an increase in proliferation and/or IL-2 production bythe T cell.

10. A polypeptide according to any one of the preceding paragraphs,wherein 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids in said amino acidsequence of B2 (ii) are substituted when compared to the amino acidsequence of SEQ ID NO: 3; optionally wherein there are no insertions ordeletions compared to the amino acid sequence of SEQ ID NO: 3.

11. A polypeptide according to paragraph 10, wherein at least one ofsaid amino acid substitutions in said amino acid sequence of said firstbinding domain is at position 122, and optionally wherein said aminoacid sequence is also substituted in at least one of positions 107, 121and 125.

12. A polypeptide according to any one of the preceding paragraphswherein said amino acid sequence of B2 comprises or consists of an aminoacid sequence selected from any one of SEQ ID NOs 8, 6, 7 and 9 to 24.

13. A polypeptide according to any one of the preceding paragraphs,wherein B1 exhibits at least one of the following functionalcharacteristics when present independently of B2:

-   -   I. binding to human OX40 with a K_(D) value which is less than        10×10⁻¹⁰M, more preferably less than 5×10⁻¹⁰M;    -   II. does not bind to murine OX40; and does not bind to other        human TNFR superfamily members, for example human CD137 or CD40

14. A polypeptide according to any one of the preceding paragraphs,wherein B1 comprises any one, two, three, four, five or all six featuresindependently selected from the following:

-   -   (a) a heavy chain CDR1 sequence which is 8 amino acids in length        and comprises the consensus sequence: “G, F, T, F, G/Y/S, G/Y/S,        Y/S, Y/S/A”;    -   (b) a heavy chain CDR2 sequence which is 8 amino acids in length        and comprises the consensus sequence: “I, G/Y/S/T, G/S/Y, S/Y,        G/S/Y, G/S/Y, G/S/Y, T”;    -   (c) a heavy chain CDR3 sequence which is 9 to 17 amino acids in        length and which comprises the consensus sequence of: “A, R,        G/Y/S/H, G/Y/F/V/D, G/Y/P/F, −/H/S, −/N/D/H, −/Y/G, −/Y, −/Y,        −/W/A/V, −/A/Y, −/D/A/Y/G/H/N, Y/S/W/A/T, UM/I/F, D, Y”    -   (d) a light chain CDR1 sequence which consists of the sequence:        “Q, S, I, S, S, Y”;    -   (e) a light chain CDR2 sequence which consists of the sequence:        “A, A, S”;    -   (f) a light chain CDR3 sequence which is 8 to 10 amino acids in        length and comprises the consensus sequence: “Q,Q, S/Y/G,        −/Y/H/G, −/S/Y/G/D, S/Y/G/D, S/Y/G/T, P/L, Y/S/H/L/F, T”;

wherein the heavy chain CDR3 sequence of (c) is preferably a sequence of10 amino acids in length which comprises the consensus sequence “A, R,Y/H, D, Y, A/Y/G, S/W/A, M/L, D, Y” or a CDR3 sequence of 11 amino acidsin length which comprises the consensus sequence “A, R, G/Y, V/F/Y, P,H, G/Y/H, Y, F/I, D, Y”; and

the light chain CDR3 sequence of (f) preferably consists of the sequence“Q, Q, S, Y, S, T, P, Y, T”.

15. A polypeptide according to any one of the preceding paragraphs,wherein B1 comprises all three heavy chain CDR sequences of a VHsequence as shown in Table A(1) and/or all three light chain CDRsequences of a VL sequence as shown in Table A(2), or wherein B1comprises a heavy chain VH sequence and/or a light chain VL sequence asshown in Table B.

16. A polypeptide according to any one of the preceding paragraphs,wherein B1 comprises a heavy chain CDR3 sequence of 11 amino acids inlength which comprises the consensus sequence “A, R, G/Y, V/F/Y, P, H,G/Y/H, Y, F/I, D, Y”; and the light chain VL sequence of SEQ ID NO: 97(1135 as shown in Table B), optionally wherein the light chain VLsequence of SEQ ID NO: 97 is present as part of the longer sequence ofSEQ ID NO: 129 (1141 as shown in Table D).

17. A polypeptide according to any one of the preceding paragraphs,wherein B1 comprises an human Fc region or a variant of a said region,where the region is an IgG1, IgG2, IgG3 or IgG4 region, preferably anIgG1 or IgG4 region.

18. A polypeptide according to any one of the preceding paragraphs,which comprises or consists of the amino acid sequence of any one of SEQID NOs 125 to 134, optionally wherein said polypeptide is a provided asa component part of an antibody.

19. A polypeptide according to any one of the preceding paragraphs foruse in a method for treating or preventing a disease or condition in anindividual.

20. A method of treating or preventing a disease or condition in anindividual, the method comprising administering to an individual apolypeptide according to any one of the preceding paragraphs.

21. A polypeptide according to paragraph 19 or a method according toparagraph 20, wherein the disease or condition is cancer and optionallywherein the individual is human.

22. A polypeptide or method according to paragraph 21, wherein themethod comprises administering the polypeptide systemically or locally,such as at the site of a tumour or into a tumour draining lymph node.

23. A polypeptide or method according to paragraph 21 or 22 wherein thecancer is prostate cancer, breast cancer, colorectal cancer, pancreaticcancer, ovarian cancer, lung cancer, cervical cancer, rhabdomyosarcoma,neuroblastoma, multiple myeloma, leukemia, acute lymphoblastic leukemia,melanoma, bladder cancer, gastric cancer, head and neck cancer, livercancer, skin cancer, lymphoma or glioblastoma.

24. A polynucleotide encoding a polypeptide according to any one ofparagraphs 1 to 18.

25. A polypeptide according to any one of paragraphs 1 to 18 conjugatedto an additional therapeutic moiety.

26. A composition comprising a polypeptide according to any one ofparagraphs 1 to 18 and at least one pharmaceutically acceptable diluentor carrier.

Further distinct embodiments are described in the following paragraphs:

1. A bispecific antibody comprising an antibody which specifically bindsto OX40 (an anti-OX40 antibody) and an antibody which specifically bindsto CD137 (an anti-CD137 antibody), joined to each other directly orindirectly.

2. A bispecific antibody according to paragraph 1 in which the CD137that is specifically bound is primate or murine, preferably human CD137,and/or wherein the OX40 that is specifically bound is primate,preferably human, OX40.

3. A bispecific antibody according to any one of the precedingparagraphs, which binds to human OX40 with a Kd of less than 50×10⁻¹⁰M,25×10⁻¹⁰M, or 20×10⁻¹⁰M and/or which binds to human CD137 with a Kdvalue which is less than 10×10⁻⁹M, 4×10⁻⁹M, or 1.2×10⁻⁹M.

4. A bispecific antibody according to any one of the precedingparagraphs, which induces an increase in the activity of an effector Tcell, optionally wherein said increase is at least 1.5 fold, 2 fold, 3fold or 5 fold higher than the increase in activity of an effector Tcell induced by a combination of the corresponding monospecificantibodies administered to the T cell as separate molecules.

5. A bispecific antibody according to paragraph 4, wherein said increasein T cell activity is an increase in proliferation and/or IL-2production and/or IFN-γ production by the T cell, optionally wherein theT cell is a CD4+ or CD8+ T cell.

6. A bispecific antibody according to any one of the precedingparagraphs, wherein the anti-OX40 antibody and the anti-CD137 antibodyare joined to each other indirectly via a linker which is a peptide ofthe amino acid sequence GGGGSGGGGSGGGGS (SEQ ID NO: 144),SGGGGSGGGGS(SEQ ID NO: 47), SGGGGSGGGGSAP (SEQ ID NO: 48), NFSQP (SEQ IDNO:49), KRTVA (SEQ ID NO: 50) or (SG)m, where m=1 to 7.

7. A bispecific antibody according to any one of the precedingparagraphs which is an scFv-IgG format bispecific antibody.

8. A bispecific antibody according to any one of the precedingparagraphs, wherein the anti-OX40 antibody exhibits at least one of thefollowing functional characteristics when present independently of theanti-CD137 antibody:

-   -   I. binding to human OX40 with a K_(D) value which is less than        10×10⁻¹⁰M, more preferably less than 5×10⁻¹⁰M;    -   II. does not bind to murine OX40; and    -   III. does not bind to other human TNFR superfamily members, for        example human CD137 or CD40

9. A bispecific antibody according to any one of the precedingparagraphs, wherein the anti-OX40 antibody comprises any one, two,three, four, five or all six features independently selected from thefollowing:

-   -   (a) a heavy chain CDR1 sequence which is 8 amino acids in length        and comprises the consensus sequence: “G, F, T, F, G/Y/S, G/Y/S,        Y/S, Y/S/A”;    -   (b) a heavy chain CDR2 sequence which is 8 amino acids in length        and comprises the consensus sequence: “I, G/Y/S/T, G/S/Y, S/Y,        G/S/Y, G/S/Y, G/S/Y, T”;    -   (c) a heavy chain CDR3 sequence which is 9 to 17 amino acids in        length and which comprises the consensus sequence of: “A, R,        G/Y/S/H, G/Y/F/V/D, G/Y/P/F, −/H/S, −/N/D/H, −/Y/G, −/Y, −/Y,        −/W/A/V, −/A/Y, −/D/A/Y/G/H/N, Y/S/W/A/T, UM/I/F, D, Y”    -   (d) a light chain CDR1 sequence which consists of the sequence:        “Q, S, I, S, S, Y”;    -   (e) a light chain CDR2 sequence which consists of the sequence:        “A, A, S”;    -   (f) a light chain CDR3 sequence which is 8 to 10 amino acids in        length and comprises the consensus sequence: “Q,Q, S/Y/G,        −/Y/H/G, −/S/Y/G/D, S/Y/G/D, S/Y/G/T, P/L, Y/S/H/L/F, T”;

wherein the heavy chain CDR3 sequence of (c) is preferably a sequence of10 amino acids in length which comprises the consensus sequence “A, R,Y/H, D, Y, A/Y/G, S/W/A, M/L, D, Y” or a CDR3 sequence of 11 amino acidsin length which comprises the consensus sequence “A, R, G/Y, V/F/Y, P,H, G/Y/H, Y, F/I, D, Y”; and

the light chain CDR3 sequence of (f) preferably consists of the sequence“Q, Q, S, Y, S, T, P, Y, T”.

10. A bispecific antibody according to any one of the precedingparagraphs, wherein the anti-OX40 antibody comprises all three heavychain CDR sequences of a VH sequence as shown in Table A(1) and/or allthree light chain CDR sequences of a VL sequence as shown in Table A(2),or wherein the anti-OX40 antibody comprises a heavy chain VH sequenceand/or a light chain VL sequence as shown in Table B.

11. A bispecific antibody according to any one of the precedingparagraphs, wherein the anti-OX40 antibody comprises a heavy chain CDR3sequence of 11 amino acids in length which comprises the consensussequence “A, R, G/Y, V/F/Y, P, H, G/Y/H, Y, F/I, D, Y”; and the lightchain VL sequence of SEQ ID NO: 97 (1135 as shown in Table B),optionally wherein the light chain VL sequence of SEQ ID NO: 97 ispresent as part of the longer sequence of SEQ ID NO: 129 (1141 as shownin Table D).

12. A bispecific antibody according to any one of the precedingparagraphs, wherein the anti-OX40 antibody when present independently ofthe anti-CD137 antibody competes for binding to OX40 with an anti-OX40antibody as defined in any one of paragraphs 8 to 11.

13. A bispecific antibody according to any one of the precedingparagraphs, wherein the anti-CD137 antibody exhibits at least one of thefollowing functional characteristics when present independently of theanti-OX40 antibody:

-   -   I. binding to human CD137 with a K_(D) value which is less than        2×10⁻⁹M, more preferably less than 1.2×10⁻⁹M; and    -   II. ability to cause an increase in activity in a CD8+ T cell in        vitro, optionally wherein said increase in activity is an        increase in proliferation and/or IL-2 production and/or IFN-γ        production by the T cell

14. A bispecific antibody according to any one of the precedingparagraphs, wherein the anti-CD137 antibody comprises all three heavychain CDR sequences of a VH sequence as shown in the first row of TableI(1) and/or all three light chain CDR sequences of a VL sequence asshown in the first row of Table I(2), or wherein the anti-CD137 antibodycomprises a heavy chain VH sequence and/or a light chain VL sequence asshown in Table H, SEQ ID NOs 177-180.

15. A bispecific antibody according to any one of the precedingparagraphs, wherein the anti-CD137 antibody when present independentlyof the anti-OX40 antibody competes for binding to CD137 with ananti-CD137 antibody as defined in paragraph 14.

16. A bispecific antibody according to any one of the precedingparagraphs, wherein the anti-OX40 and/or the anti-CD137 antibodycomprises an human Fc region or a variant of a said region, where theregion is an IgG1, IgG2, IgG3 or IgG4 region, preferably an IgG1 or IgG4region.

17. A bispecific antibody according to any one of the precedingparagraphs, which comprises the amino acid sequence of any one of SEQ IDNOs 149, 151, 153, 155, 157, 159, 161 or 163.

18. A bispecific antibody according to any one of the precedingparagraphs which comprises the amino acid sequence of any one of SEQ IDNos 165, 167, 169, 171 or 173.

19. A bispecific antibody according to any one of the precedingparagraphs which comprises the amino acid sequences of:

-   -   SEQ ID NO: 165 and any one of SEQ ID NOs: 149, 153, 159 and 163;        or    -   SEQ ID NO: 167 and SEQ ID NO 151; or    -   SEQ ID NO: 169 and SEQ ID NO: 155; or    -   SEQ ID NO: 171 and SEQ ID NO: 157; or    -   SEQ ID NO: 173 and SEQ ID NO: 161.

20. A bispecific antibody according to any one of the precedingparagraphs for use in a method for treating or preventing a disease orcondition in an individual.

21. A method of treating or preventing a disease or condition in anindividual, the method comprising administering to an individual abispecific antibody according to any one of the preceding paragraphs.

22. A bispecific antibody according to paragraph 20 or a methodaccording to paragraph 21 wherein the disease or condition is cancer andoptionally wherein the individual is human.

23. A bispecific antibody or method according to paragraph 22, whereinthe method comprises administering the bispecific antibody systemicallyor locally, such as at the site of a tumour or into a tumour draininglymph node.

24. A bispecific antibody or method according to paragraph 22 or 23wherein the cancer is prostate cancer, breast cancer, colorectal cancer,pancreatic cancer, ovarian cancer, lung cancer, cervical cancer,rhabdomyosarcoma, neuroblastoma, multiple myeloma, leukemia, acutelymphoblastic leukemia, melanoma, bladder cancer, gastric cancer, headand neck cancer, liver cancer, skin cancer, lymphoma or glioblastoma.

25. A polynucleotide encoding at least one polypeptide chain of abispecific antibody according to any one of paragraphs 1 to 20.

26. A composition comprising a bispecific antibody according to any oneof paragraphs 1 to 20 and at least one pharmaceutically acceptablediluent or carrier.

27. An antibody specific for CD137 which is as defined in any one ofparagraphs 13 to 15.

BRIEF DESCRIPTION OF THE FIGURES

Preferred, non-limiting examples which embody certain aspects of theinvention will now be described, with reference to the followingfigures:

FIG. 1 shows a schematic representation of the structure of exemplaryarrangements for the bispecific polypeptides of the invention. Anti-OX40antibody variable domains are filled in black; constant domains inwhite. CTLA-A binding domains are shaded with diagonal lines.

FIG. 2 shows the CTLA-4 binding properties of CTLA-4 binding domains ofpolypeptides the invention as determined by an ELISA binding assay.

FIG. 3 shows the CTLA-4 binding properties of CTLA-4 binding domains ofpolypeptides of the invention as determined by an ELISA inhibitionassay.

FIG. 4 provides a schematic representation of human wild-type CD86 aminoacid sequences disclosed herein. (A) is the amino acid sequence of themonomeric soluble extracellular domain of human CD86 without N-terminalsignal sequence (SEQ ID NO: 3); (B) is the amino acid sequence of themonomeric extracellular and transmembrane domains of human wildtypeCD86, including N-terminal signal sequence (SEQ ID NO: 4); (C) is thefull length amino acid sequence of human CD86 (Genbank ABK41931.1; SEQID NO: 44). The sequence in A may optionally lack Alanine and Proline atthe N terminus, i.e. positions 24 and 25, shown in bold. Signalsequences in B and C are underlined. Numbering of amino acid positionsis based on SEQ ID NOs: 4 and 44, starting from the N terminus.

FIG. 5 shows the results of an inhibition ELISA demonstrating that aCTLA-4 binding domains of polypeptides of the invention has bindingaffinity of a similar magnitude for both human and murine CTLA-4.

FIG. 6 is a plot of dissociation rate constant versus association rateconstant for exemplary anti-OX40 antibodies, as determined by surfaceplasmon resonance.

FIG. 7 shows binding of exemplary anti-OX40 antibodies to human OX40overexpressed on CHO cells, measured by flow cytometry.

FIG. 8 shows the level of IL-2 production by T cells when incubated invitro with different exemplary anti-OX40 antibodies. The y-axis is theratio of the top value of IL-2 production by a tested antibody/the topvalue of a reference antibody. Mean and SEM values from at least 4donors are shown.

FIG. 9 shows results of an ELISA assay for binding of exemplarybispecific molecules to individual targets OX40 and CTLA4.

FIG. 10 shows results of surface plasmon resonance analysis of bindingof exemplary bispecific molecules to both OX40 and CTLA4. The differentbispecific antibodies were passed over the sensor (start indicated byI)). At near saturation of the surface, buffer was applied (II) andsubsequently CTLA-4 (III) was passed over the sensor surface generatinga second association phase, represented by the full line. After threeminutes, buffer (IV) was applied, and the following dissociation phasereflects dissociation of both CTLA-4 and OX40 Ab. As a control, onlybuffer, with no CTLA-4 was added, represented by the dotted line.

FIG. 11 shows results of an ELISA assay showing binding of exemplarybispecific molecules to both OX40 and CTLA-4 simultaneously.

FIG. 12 shows the level of IL-2 production by T cells when incubated invitro with different exemplary bispecific molecules in a titrationseries: A) 1164/1141 and 1166/1141 B) 1168/1141 and 1170/1263 C)1514/1581 and 1520/1141 D) 1526/1585 and 1542/1141 or a combination ofthe two corresponding monospecific antibodies for each target(monoclonal OX40 antibodies or the CTLA-4-binding domain coupled to anisotype IgG antibody: 1756/1757). The assay was performed in U-shapednon-tissue cultured treated 96-well plates coated with CD3 (UCHT1) andCTLA-4 (Orencia). Mean out of 4 donors is presented.

FIG. 13 shows the level of IL-2 production by T cells when incubated invitro with different exemplary bispecific molecules at 1.49 nM or acombination of the corresponding monospecific antibodies for each target(a-OX40 mAbs or the CTLA-4− domain coupled to an isotype antibody:1756/1757). The assay was performed in U-shaped non-tissue culturedtreated 96-well plates coated with anti-CD3 (UCHT1) with or withoutCTLA-4 (Orencia), indicated by + or −. Mean and SD out of 4 donors ispresented.

FIG. 14 shows binding of exemplary anti-CD137 antibody 1204/1205 tohuman and cynomolgus monkey CD137 overexpressed on CHO cells, measuredby flow cytometry.

FIG. 15 shows the normalized stimulation index of IFNγ production ofexemplary anti-CD137 antibody 1204/1205 compared with reference antibody111/112 in a CD8 T cell agonist assay.

FIG. 16 shows the level of IL-2 production by T cells when incubated invitro with different exemplary bispecific molecules in a titrationseries: A) 1164/1135-1204/1205. B) 1166/1167-1204/1205, C)1168/1135-1204/1205, D) 1170/1171-1204/1205, E) 1482/1483-1204/1205, F)1520/1135-1204/1205, G) 1526/1527-1204/1205, H) 1542/1135-1204/1205; ora combination of the two corresponding monospecific antibodies for eachtarget. An isotype control 1188/1187 (IgG1) is used as a negativecontrol in all experiments. The cells were cultured for 72 h in U-shapednon-tissue cultured treated 96-well plates coated with anti-CD3 antibody(UCHT1). Mean IL-2 levels of 2 donors is presented.

FIG. 17 shows the level of IL-2 production by peripheral bloodmononuclear cells when incubated in vitro with exemplary bispecificmolecule 1164/1135-1204/1205 at 1 nM, compared to the correspondingmonospecific antibodies for each target: anti-OX40 mAb 1164/1135 or mAbanti-CD137 1204/1205 or an isotype control 1188/1187 (IgG1). The cellswere cultured for 48 h in U-shaped non-tissue cultured treated 96-wellplates coated with anti-CD3 antibody (UCHT1). Mean IL-2 levels of 2donors is presented.

FIG. 18 shows results of an ELISA assay showing binding of exemplarybispecific molecules to both OX40 and CD137 simultaneously.

FIG. 19 shows a schematic representation of the structure of exemplaryformats for bispecific antibodies of the invention. In each format, theconstant regions are shown as filled light grey; variable heavy chainregions VH1 are shown as checkered black and white; variable light chainregions VL1 are shown as filled white; variable heavy chain regions VH2are shown as filled black; and variable light chain regions VL2 areshown as white with diagonal lines. OX40 binding domains (bindingdomain 1) are typically represented as a pair of a checkered black andwhite domain with a filled white domain (VH1/VL1); CD137 binding domains(binding domain 2) are typically represented as a pair of a filled blackdomain and a white domain with diagonal lines (VH2/VL2). However, in allof the formats shown, it will be appreciated that binding domains 1 and2 may be switched. That is, an OX40 binding domain may occur in aposition shown in this figure for a CD137 domain, and vice versa.Furthermore, binding domain 2 may occur in different variable heavy andlight chain orders, i.e. either in VH2/VL2 or VL2/VH2 order.

FIG. 20 shows binding of the different CD137 antibodies to human CD137and cynomolgus CD137 expressed on cells and measured by flow cytometry.

FIG. 21 shows an overview of human/mouse CD137 chimeras (mousesequence=black lines, human sequence=white lines).

FIG. 22 shows the results from an example were the ability to blockCD137L binding was evaluated. 100% binding reflects no blocking. Signalsbelow 100% reflects blocking of the binding (antibody concentration fromleft to right 0.25, 2.5 and 25 μg/ml).

FIG. 23 shows the ability of monospecific CD137 antibodies to stimulateCD8 T cells as measured by IFNγ release.

FIG. 24 shows the results from a dual ELISA. CD137 was coated to anELISA well and the bispecific antibody was added at differentconcentrations. Binding was detected using biotinylated CTLA-4.

FIG. 25 shows IFNγ levels in supernatants measured from human CD8positive T cells stimulated with 1.5 nM CD137-CTLA4 bispecificantibodies or an isotype control with CTLA-4 binding only. The assay wasperformed on plates coated with CD3 with or without CTLA-4.

FIG. 26 shows full dilution curve of IFNγ levels in supernatantsmeasured from human CD8 positive T cells stimulated with CD137-CTLA4bispecific antibodies (at different concentrations) or an isotypecontrol with CTLA-4 binding only. The assay was performed on platescoated with CD3 with CTLA-4.

FIG. 27 shows induction of ADCC at different concentration bymonospecific CTLA-4 (Control IgG with CTLA-4 binding part, i.e. domain)and OX40 (1166/1167) binding molecules, alone and in combination,compared to ADCC induced by an exemplary bispecific antibody targetingCTLA-4 and OX40.

FIG. 28. CHO cells expressing both CTLA-4 and OX40 were stained withdecreasing concentrations of 1166/1261, or the two monospecific binders1166/1167 (OX40 specific monoclonal antibody) and control IgG withCTLA-4 binding part (monospecific CTLA4 binding IgG fusion protein) (200nM-0,0034 nM), followed by PE-conjugated anti-human IgG. Fluorescencewas detected using flow cytometry. ‘Ctr IgG’ is a negative isotypecontrol.

FIG. 29. HEK-CTLA4 and CHO-OX40 were stained with PKH26 and PKH67respectively and incubated with 1166/1261 or a combination of the twomonospecific OX40 and CTLA-4 binding molecules (1166/1167 and ControlIgG with CTLA-4 binding part). The percentage ofdouble-positive/aggregated cells were quantified using flow cytometry(representative experiment).

FIG. 30. Plasma levels of the bispecific OX40-CTLA-4 antibody 1166/1261and the monospecific OX40 antibody 1166/1167 measured at different timepoints following administration. Two different ELISA methods were used,ELISA-1, where OX40 was coated on the wells and anti-Fc was used todetect binding and ELISA-2, where OX40 was coated on the wells andbiotinylated CTLA-4 was used for detection.

FIG. 31. HT-29 colon carcinoma cells (4×10⁶) were inoculatedsubcutaneously to the right hind flank/back at day 0. Human PBMC cells(7×10⁶) were administered intraperitoneally on the same day. Thetreatments were done by intraperitoneal injections (667 nmol/dose) ondays 6, 13, and 20. N(mice)=5/donor, n(donor=4), pooled data from HT29responders.

DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 is the amino acid sequence of human CTLA-4 (correspondingto GenBank: AAD00698.1)

SEQ ID NO: 2 is the amino acid sequence of human CD28 (corresponding toGenBank: AAA51944.1)

SEQ ID NO: 3 is the amino acid sequence of the monomeric extracellulardomain of human wildtype CD86, excluding a 23 amino acid signal sequencefrom the N terminus.

SEQ ID NO: 4 is the amino acid sequence of the monomeric extracellularand transmembrane domains of human wildtype CD86, including N-terminalsignal sequence (see FIG. 4). All numbering of amino acid positionsherein is based on the positions in SEQ ID NO: 4 starting from the Nterminus. Thus, the Alanine at the N terminus of SEQ ID NO: 3 isnumbered 24.

SEQ ID NO: 5 is the amino acid sequence of a mutant form of theextracellular domain of human CD86 disclosed in Peach et al (Journal ofBiological Chemistry 1995, vol 270(36), 21181-21187). H at position 79of the wild type sequence is substituted with A in the correspondingposition for the sequence of SEQ ID NO: 5. This change is referred toherein as H79A. Equivalent nomenclature is used throughout for otheramino acid substitutions referred to herein. Numbering of positions isbased on SEQ ID NO: 4 as outlined above.

SEQ ID NOs: 6 to 24 are the amino acid sequences of specific proteins ofthe invention.

SEQ ID NOs: 25 to 43 are nucleotide sequences encoding the amino acidsequences of each of SEQ ID NOs 6 to 24, respectively

SEQ ID NO: 44 is the full length amino acid sequence of human CD86(corresponding to GenBank: ABK41931.1)

SEQ ID NO: 45 is the amino acid sequence of murine CTLA-4 (correspondingto UniProtKB/Swiss-Prot: P09793.1).

SEQ ID NO: 46 is the amino acid sequence of murine CD28 (correspondingto GenBank: AAA37395.1).

SEQ ID NOs: 47 to 50 are various linkers which may be used in thebispecific polypeptides of the invention.

SEQ ID NO: 51 is the amino acid sequence of human OX40 (corresponding toGenBank: NP_003318.1)

SEQ ID NOs: 52 to 88 are exemplary CDR sequences of anti-OX40 antibodiesdisclosed herein.

SEQ ID NOs: 89 to 124 are exemplary amino acid and nucleotide sequencesof the heavy and light chain variable regions of antibodies disclosedherein.

SEQ ID NOs: 125 to 134 are exemplary amino acid and nucleotide sequencesof bispecific polypeptides disclosed herein.

SEQ ID NO: 135 is an exemplary heavy chain constant region amino acidsequence.

SEQ ID NO: 136 is an exemplary light chain constant region amino acidsequence.

SEQ ID NO: 137 is an exemplary modified human heavy chain IgG4 constantregion sequence with a mutation from Ser to Pro in the hinge region(position 108) and from His to Arg in the CH3 region (position 315).Mutations result in reduced serum half-life and stabilization of thecore hinge of IgG4 making the IgG4 more stable, preventing Fab armexchange.

SEQ ID NO: 138 is an exemplary wild type human heavy chain IgG4 constantregion sequence. That is a sequence lacking the mutations of SEQ ID NO:137.

SEQ ID NO: 139 is an exemplary modified human heavy chain IgG4 constantregion sequence with a single mutation from Ser to Pro in the hingeregion (position 108). Mutation results in stabilization of the corehinge of IgG4 making the IgG4 more stable, preventing Fab arm exchange.

SEQ ID NO: 140 is an exemplary cDNA sequence (i.e. lacking introns)encoding the IgG4 constant region of SEQ ID NO: 137.

SEQ ID NO: 141 is an exemplary genomic DNA sequence (i.e. includingintrons) encoding the IgG4 constant region of SEQ ID NO: 137

SEQ ID NO: 142 is an exemplary cDNA sequence (i.e. lacking introns)encoding the IgG4 constant region of SEQ ID NO: 138.

SEQ ID NO: 143 is an exemplary genomic DNA sequence (i.e. includingintrons) encoding the IgG4 constant region of SEQ ID NO: 138.

SEQ ID NO 144 is a linker which may be used in the bispecificpolypeptides of the invention.

SEQ ID NOs: 145 and 146 are exemplary cDNA and genomic DNA sequences,respectively, encoding the IgG1 constant region of SEQ ID NO: 135.

SEQ ID NOs: 147 is an exemplary DNA sequence encoding the light chainkappa region of SEQ ID NO: 136.

SEQ ID NO: 148 is the amino acid sequence of human CD137 (correspondingto GenBank: AAH06196.1)

SEQ ID NOs: 149 to 174 are amino acid and nucleotide sequences ofexemplary bispecific antibodies disclosed herein.

SEQ ID NO: 175 is an exemplary cDNA sequence (i.e. lacking introns)encoding the IgG4 region of SEQ ID NO: 139.

SEQ ID NO: 176 is an exemplary genomic DNA sequence (i.e. includingintrons) encoding the IgG4 region of SEQ ID NO: 139.

SEQ ID NOs: 177 to 196 are exemplary amino acid and nucleotide sequencesof the heavy and light chain variable regions of anti-CD137 antibodiesdisclosed herein.

SEQ ID NOs: 197 to 206 are exemplary bispecific polypeptides of theinvention.

SEQ ID NOs 207 to 226 correspond to exemplary CDR sequences for CD137binding domains (as do SEQ ID NOs 80 and 81)

Tables (Sequences)

TABLE A(1) Exemplary heavy chain CDR sequences (OX40 antibody) VH numberSEQ H CDR1 SEQ H CDR2 SEQ H CDR3 1166 52 GFTFGGYY 60 ISGSGGST 69ARYDYASMDY 1170 As 1166 61 IPGSGGST 70 ARYDYYWMDY 1164 58 GFTFYGSS 62IYSSGGYT 71 ARGVPHGYFDY 1168 54 GFTFSGSS 63 ISYYGGYT 72 ARYFPHYYFDY 148255 GFTFSSYA 64 ISYYSGYT 73 ARGYGYLDY 1490 As 1482 As 1168 74 ARYYPHHYIDY1514 56 GFTFGYYY 65 ISSYGSYT 75 ARSGYSNWANS FDY 1520 As 1482 As 1166 76ARYYYSHGYYV YGTLDY 1524 57 GFTFGSYY 66 IGSYYGYT 77 ARHDYGALDY 1526 58GFTFSGYS 67 IGYSGYGT 78 ARYYFHDYAAYS LDY 1542 59 GFTFGSSS 68 IGYYSYST 79ARGYPHHYFDY S

TABLE A(2) Exemplary light chain CDR sequences (OX40 antibody) VL numberSEQ L CDR1 SEQ L CDR2 SEQ L CDR3 1167 80 QSISSY 81 AAS 82 QQYYWYGLST1171 As 1167 As 1167 83 QQGHGSYPHT 1135 As 1167 As 1167 84 QQSYSTPYT1483 As 1167 As 1167 85 QQYGSLLT 1515 As 1167 As 1167 86 QQGDYTLFT 1525As 1167 As 1167 87 QQYGPSGLFT 1527 As 1167 As 1167 88 QQYGSDSLLT

TABLE B Exemplary sequences (OX40 antibody) SEQ ID NO. CHAIN NO. TYPESEQUENCE 89 1167, light chain aa DIQMTQSPSSLSASVGDRVTITCRASQSISSYLN VLWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQYYWYGLSTFGQGTKLEIK 90 1167, light chain nt GACATCCAGATGACCCAGTCTCCATCCTCCC VLTGAGCGCATCTGTAGGAGACCGCGTCACCAT CACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAA AGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCA GTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG CAACTTATTACTGTCAACAGTACTACTGGTACGGTCTGTCCACTTTTGGCCAGGGGACCAAGC TGGAGATCAAA 91 1166, heavy aaEVQLLESGGGLVQPGGSLRLSCAASGFTFGGY chain VHYMSWVRQAPGKGLEWVSAISGSGGSTYYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYDYASMDYWGQGTLVTVSS 92 1166, heavy nt GAGGTGCAGCTGTTGGAGAGCGGGGGAGGCchain VH TTGGTACAGCCTGGGGGGTCCCTGCGCCTCT CCTGTGCAGCCAGCGGATTCACCTTTGGTGGTTACTACATGTCTTGGGTCCGCCAGGCTCCA GGGAAGGGGCTGGAGTGGGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTATGCAGA CTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAAT GAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTACGACTACGCTTCTA TGGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 93 1171, light chain aa DIQMTQSPSSLSASVGDRVTITCRASQSISSYLN VLWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQGHGSYPHTFGQGTKLEIK 94 1171, light chain nt GACATCCAGATGACCCAGTCTCCATCCTCCC VLTGAGCGCATCTGTAGGAGACCGCGTCACCAT CACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAA AGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCA GTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG CAACTTATTACTGTCAACAGGGTCATGGTTCTTACCCGCACACTTTTGGCCAGGGGACCAAGC TGGAGATCAAA 95 1170, heavy aaEVQLLESGGGLVQPGGSLRLSCAASGFTFGGY chain VHYMSWVRQAPGKGLEWVSYIPGSGGSTYYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYDYYWMDYWGQGTLVTVSS 96 1170, heavy nt GAGGTGCAGCTGTTGGAGAGCGGGGGAGGCchain VH TTGGTACAGCCTGGGGGGTCCCTGCGCCTCT CCTGTGCAGCCAGCGGATTCACCTTTGGTGGTTACTACATGTCTTGGGTCCGCCAGGCTCCA GGGAAGGGGCTGGAGTGGGTCTCATACATTCCTGGTTCTGGTGGTTCTACATACTATGCAGAC TCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATG AACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTACGACTACTACTGGATG GACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 97 1135, light chain aa DIQMTQSPSSLSASVGDRVTITCRASQSISSYLN VLWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQSYSTPYTFGQGTKLEIK 98 1135, light chain nt GACATCCAGATGACCCAGTCTCCATCCTCCC VLTGAGCGCATCTGTAGGAGACCGCGTCACCAT CACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAA AGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCA GTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG CAACTTATTACTGTCAACAGAGTTACAGTACCCCTTATACTTTTGGCCAGGGGACCAAGCTGG AGATCAAA 99 1164, heavy aaEVQLLESGGGLVQPGGSLRLSCAASGFTFYGS chain VHSMYWVRQAPGKGLEWVSGIYSSGGYTSYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGVPHGYFDYWGQGTLVTVSS 100 1164, heavy nt GAGGTGCAGCTGTTGGAGAGCGGGGGAGGCchain VH TTGGTACAGCCTGGGGGGTCCCTGCGCCTCT CCTGTGCAGCCAGCGGATTCACCTTTTACGGTTCTTCTATGTACTGGGTCCGCCAGGCTCCA GGGAAGGGGCTGGAGTGGGTCTCAGGTATTTACTCTTCTGGTGGTTACACATCTTATGCAGAC TCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATG AACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCGGTGTTCCTCATGGTTAC TTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 101 1168, heavy aa EVQLLESGGGLVQPGGSLRLSCAASGFTFSGS chain VHSMSWVRQAPGKGLEWVSSISYYGGYTYYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYFPHYYFDYWGQGTLVTVSS 102 1168, heavy nt GAGGTGCAGCTGTTGGAGAGCGGGGGAGGCchain VH TTGGTACAGCCTGGGGGGTCCCTGCGCCTCT CCTGTGCAGCCAGCGGATTCACCTTTAGTGGTTCTTCTATGTCTTGGGTCCGCCAGGCTCCA GGGAAGGGGCTGGAGTGGGTCTCATCTATTTCTTACTACGGTGGTTACACATACTATGCAGAC TCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATG AACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTACTTCCCGCATTACTAC TTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 103 1483, light chain aa DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNVL WYQQKPGKAPKLLIYAASSLQSGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQYGSLLTFGQGTKLEIK 104 1483, light chain nt GACATCCAGATGACCCAGTCTCCATCCTCCC VLTGAGCGCATCTGTAGGAGACCGCGTCACCAT CACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAA AGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCA GTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG CAACTTATTACTGTCAACAGTACGGTTCTCTGCTCACTTTTGGCCAGGGGACCAAGCTGGAGA TCAAA 105 1482, heavy aaEVQLLESGGGLVQPGGSLRLSCAASGFTFSSY chain VHAMSWVRQAPGKGLEWVSYISYYSGYTYYADSV KGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYGYLDYWGQGTLVTVSS 106 1482, heavy nt GAGGTGCAGCTGTTGGAGAGCGGGGGAGGCchain VH TTGGTACAGCCTGGGGGGTCCCTGCGCCTCT CCTGTGCAGCCAGCGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCA GGGAAGGGGCTGGAGTGGGTCTCATACATTTCTTACTACTCTGGTTACACATACTATGCAGAC TCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATG AACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCGGTTACGGTTACTTGGA CTATTGGGGCCAGGGAACCCTGGTCACCGTC TCCTCA107 1490, heavy aa EVQLLESGGGLVQPGGSLRLSCAASGFTFSSY chain VHAMSWVRQAPGKGLEWVSGISYYGGYTYYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYPHHYIDYWGQGTLVTVSS 108 1490, heavy nt GAGGTGCAGCTGTTGGAGAGCGGGGGAGGCchain VH TTGGTACAGCCTGGGGGGTCCCTGCGCCTCT CCTGTGCAGCCAGCGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCA GGGAAGGGGCTGGAGTGGGTCTCAGGTATTTCTTACTACGGTGGTTACACATACTATGCAGAC TCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATG AACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTACTACCCGCATCATTAC ATTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 109 1515, light chain aa DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNVL WYQQKPGKAPKLLIYAASSLQSGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQGDYTLFTFGQGTKLEIK 110 1515, light chain nt GACATCCAGATGACCCAGTCTCCATCCTCCC VLTGAGCGCATCTGTAGGAGACCGCGTCACCAT CACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAA AGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCA GTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG CAACTTATTACTGTCAACAGGGTGATTACACTCTGTTCACTTTTGGCCAGGGGACCAAGCTGG AGATCAAA 111 1514, heavy aaEVQLLESGGGLVQPGGSLRLSCAASGFTFGYY chain VHYMSWVRQAPGKGLEWVSGISSYGSYTYYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSGYSNWANSFDYWGQGTLVTVSS 112 1514, heavy ntGAGGTGCAGCTGTTGGAGAGCGGGGGAGGC chain VH TTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTGGTTA CTACTACATGTCTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTT CTTCTTACGGTAGTTACACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTG ACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTAT ATTATTGTGCGCGCTCTGGTTACTCTAACTGGGCTAACTCTTTTGACTATTGGGGCCAGGGAA CCCTGGTCACCGTCTCCTCA 113 1520, heavy aaEVQLLESGGGLVQPGGSLRLSCAASGFTFSSY chain VHAMSWVRQAPGKGLEWVSAISGSGGSTYYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYYSHGYYVYGTLDYWGQGTLVTVSS 114 1520, heavy ntGAGGTGCAGCTGTTGGAGAGCGGGGGAGGC chain VH TTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTAGCAG CTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTA GTGGTAGTGGTGGTAGCACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGT GACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGT ATATTATTGTGCGCGCTACTACTACTCTCATGGTTACTACGTTTACGGTACTTTGGACTATTGG GGCCAGGGAACCCTGGTCACCGTCTCCTCA 1151525, light chain aa DIQMTQSPSSLSASVGDRVTITCRASQSISSYLN VLWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQYGPSGLFTFGQGTKLEIK 116 1525, light chain nt GACATCCAGATGACCCAGTCTCCATCCTCCC VLTGAGCGCATCTGTAGGAGACCGCGTCACCAT CACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAA AGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCA GTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG CAACTTATTACTGTCAACAGTACGGTCCGTCTGGTCTGTTCACTTTTGGCCAGGGGACCAAGC TGGAGATCAAA 117 1524, heavy aaEVQLLESGGGLVQPGGSLRLSCAASGFTFGSY chain VHYMGWVRQAPGKGLEWVSSIGSYYGYTYYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHDYGALDYWGQGTLVTVSS 118 1524, heavy nt GAGGTGCAGCTGTTGGAGAGCGGGGGAGGCchain VH TTGGTACAGCCTGGGGGGTCCCTGCGCCTCT CCTGTGCAGCCAGCGGATTCACCTTTGGTTCTTACTACATGGGTTGGGTCCGCCAGGCTCCA GGGAAGGGGCTGGAGTGGGTCTCATCTATTGGTTCTTACTACGGTTACACATACTATGCAGAC TCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATG AACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCCATGACTACGGTGCTTT GGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 119 1527, light chain aa DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNVL WYQQKPGKAPKLLIYAASSLQSGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQYGSDSLLTFGQGTKLEIK 120 1527, light chain nt GACATCCAGATGACCCAGTCTCCATCCTCCC VLTGAGCGCATCTGTAGGAGACCGCGTCACCAT CACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAA AGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCA GTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTG CAACTTATTACTGTCAACAGTACGGTTCTGATTCTCTGCTCACTTTTGGCCAGGGGACCAAGC TGGAGATCAAA 121 1526, heavy aaEVQLLESGGGLVQPGGSLRLSCAASGFTFSGY chain VHSMYWVRQAPGKGLEWVSGIGYSGYGTYYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARYYFHDYAAYSLDYWGQGTLVTVSS 122 1526, heavy ntGAGGTGCAGCTGTTGGAGAGCGGGGGAGGC chain VH TTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTGG TTACTCTATGTACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATT GGTTACTCTGGTTACGGTACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGT GACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGT ATATTATTGTGCGCGCTACTACTTCCATGACTACGCTGCTTACTCTTTGGACTATTGGGGCCA GGGAACCCTGGTCACCGTCTCCTCA 1231542, heavy aa EVQLLESGGGLVQPGGSLRLSCAASGFTFGSS chain VHSMYWVRQAPGKGLEWVSGIGYYSYSTSYADS VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGYPHHYFDYWGQGTLVTVSS 124 1542, heavy nt GAGGTGCAGCTGTTGGAGAGCGGGGGAGGCchain VH TTGGTACAGCCTGGGGGGTCCCTGCGCCTCT CCTGTGCAGCCAGCGGATTCACCTTTGGTTCTTCTTCTATGTACTGGGTCCGCCAGGCTCCA GGGAAGGGGCTGGAGTGGGTCTCAGGTATTGGTTACTACTCTTACTCTACATCTTATGCAGA CTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAAT GAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCGGTTACCCGCATCATT ACTTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

TABLE C Exemplary variants of domain of human C086 SEQ ID NO.DESIGNATION SEQUENCE 6 900 LKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVDSKYMGRTSFDSDSWTLRLHNLQIKDKGIYQCVIHHK KPSGLVKIHEMNSELSVLA 7 901LKIQAYFNETADLPCQFANSQNLTLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGIYQCVIHHKKP TGMIKIHEMNSELSVLT 8 904LKIQAYFNETADLPCQFANSQNQSLSELIVFWQDQENLVLNEVYLGKERFDAVDSKYMGRTSFDSDSWTLRLHNLQIKDKGIYQCIIHHKKP SGMVKIHQMDSELSVLA 9 906LKIQAYINETADLPCQFANSQNLSLSELVVFWQDQENLVLNEVYLGKERFDSVDSKYMGRTSFDSDSWTLRLHNLQIKDKGFYQCIIHHKKP TGLVKIHEMNSELSVLA 10 907LKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKK PTGMIKIHEMNSELSVLA 11 908LKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGIYQCIIHHKK PTGMVKIHEMNSELSVLA 12 910LKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVDSKYMGRTSFDSDSWTLRLHNLQIKDKGIYQCIIHHKK PTGMVKIHEMNSELSVLA 13 915LKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLILNEVYLGKEKFDSVDSKYMGRTSFDSDSWTLRLHNLQIKDKGFYQCIIHHKKP SGLIKIHQMDSELSVLA 14 938LKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLILNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGIYQCIIHHKKP TGMVKIHQMNSELSVLA 15 1038APLKIQAYFNETADLPCQFANSQNLSLSELVVFWQDQENLVLNEVYLGKEKFDSVDSKYMGRTSFDSDSWTLRLHNLQIKDKGIYQCIIHHK KPTGMVKIHEMNSELSVLA 161039 APLKIQAYFNETADLPCQFANSQNLSLSELVVFWQDQENLVLNEVYLGKEKFDSVSSKYMGRTSFDSDSWTLRLHNLQIKDKGIYQCIIHHK KPSGMVKIHQMDSELSVLA 171040 APLKIQAYFNETADLPCQFANSQNLSLSELVVFWQDQENLVLNEVYLGKERFDSVDSKYMGRTSFDSDSWTLRLHNLQIKDKGRYQCIIHH KKPTGMINIHQMNSELSVLA 181041 APLKIQAYLNETADLPCQFANSQNLSLSELVVFWQDQENLVLNEVYLGKEKFDSVDSKYMGRTSFDSDSWTLRLHNLQIKDKGIYQCIIHHK KPTGLVKIHEMNSELSVLA 191042 APLKIQAYFNETADLPCQFANSQNLSLSELVVFWQDQENLVLNEVYLGKEIFDSVSSKYMGRTSFDSDSWTLRLHNLQIKDKGIYQCIIHHKK PSGMVKIHQMDSELSVLA 201043 APLKIQAYFNETADLPCQFANSQNLSLSELVVFWQDQENLVLNEVYLGKEKFDSVDSKYMGRTSFDSDSWTLRLHNLQIKDKGIYQCIIHHK KPTGMIKIHEMNSELSVLA 211044 APLKIQAYFNETADLPCQFANSQNLTLSELVVFWQDQENLVLNEVYLGKEKFDSVSSKYMGRTSFDSDSWTLRLHNLQIKDKGIYQCIIHHK KPTGMIKIHEMSSELSVLA 221045 APLKIQAYFNETADLPCQFANSQNLTLSELVVFWQDQENLVLNEVYLGKEKFDSVDSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHK KPTGLVKIHEMNSELSVLA 231046 APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVDSKYMGRTSFDSDSWTLRLHNLQIEDKGIYQCIIHH KKPSGMVKIHQMDSELSVLA 241047 APLKIQAYFNETADLPCQFANSQNLSLSELVVFWQDQENLVLNEVYLGKEKFDSVDSKYMGRTSFDSDSWTLRLHNLQIKDKGIYQCIIHHK KPTGLVKIHEMNSELSVLA

TABLE D Exemplary polypeptides for OX40 and CTLA-4 SEQ ID NO.DESIGNATION TYPE SEQUENCE 125 1261 = aaDIQMTQSPSSLSASVGDRVTITCRASQSISSYLN 1167 light chainWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSG VL, withSGTDFTLTISSLQPEDFATYYCQQYYWYGLSTF constant kappaGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV sequence, linkerVCLLNNFYPREAKVQWKVDNALQSGNSQESVT (underlined) andEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT CD86 mutantHQGLSSPVTKSFNRGECSGGGGSGGGGSAPL 1040 KIQAYFNETADLPCQFANSQNLSLSELVVFWQDQENLVLNEVYLGKERFDSVDSKYMGRTSFDSD SWTLRLHNLQIKDKGRYQCIIHHKKPTGMINIHQMNSELSVLA LIGHT CHAIN PREFERABLY ASSEMBLESWITH A HEAVY CHAIN COMPRISING THE 1166 VH SEQUENCETHUS, COMPLETE MOLECULE MAY BE DESIGNATED 1166/1261 126 1261 = 1267 ntGACATCCAGATGACCCAGTCTCCATCCTCCC light chain VL,TGAGCGCATCTGTAGGAGACCGCGTCACCAT with constantCACTTGCCGGGCAAGTCAGAGCATTAGCAGC kappa TATTTAAATTGGTATCAGCAGAAACCAGGGAAsequence, linker AGCCCCTAAGCTCCTGATCTATGCTGCATCC and CD86AGTTTGCAAAGTGGGGTCCCATCACGTTTCA mutant 1040GTGGCAGTGGAAGCGGGACAGATTTCACTCT CACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACTACTGGTAC GGTCTGTCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACgtgagtcgtacgctagcaagcttgatatcgaattctaaactctgagggggtcggatgacgtggccattctttgcctaaagcattgagtttactgcaaggtcagaaaagcatgcaaagccctcagaatggctgcaaagagctccaacaaaacaatttagaactttattaaggaatagggggaagctaggaagaaactcaaaacatcaagattttaaatacgcttcttggtctccttgctataattatctgggataagcatgctgttttctgtctgtccctaacatgccctgtgattatccgcaaacaacacacccaagggcagaactttgttacttaaacaccatcctgtttgcttctttcctcagGAACTGTGGCTGCACCATCTGTOTTCA TCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATA ACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAAC TCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCT GACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGG GCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTAGCGGAGGAGGAGGAAG CGGAGGAGGAGGAAGCGCCCCCCTCAAAATCCAAGCGTACTTCAACGAAACTGCAGACTTA CCGTGTCAGTTTGCCAATTCGCAGAATCTGAGCCTGAGCGAACTGGTGGTTTTCTGGCAGGA TCAGGAGAACCTGGTTCTGAACGAAGTCTATCTGGGCAAAGAGCGGTTCGACAGCGTGGAC AGCAAGTATATGGGCCGCACCAGCTTTGATAGCGACAGCTGGACCCTGCGTCTGCACAATCT GCAAATCAAAGATAAGGGTAGGTACCAGTGCATTATCCACCATAAGAAGCCGACGGGTATGA TTAATATTCACCAAATGAACTCCGAGTTGTCTGTCCTGGCG 127 1263 = 1171 aa DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNlight chain VL, WYQQKPGKAPKLLIYAASSLQSGVPSRFSGSG with constantSGTDFTLTISSLQPEDFATYYCQQGHGSYPHTF kappaGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV sequence, linkerVCLLNNFYPREAKVQWKVDNALQSGNSQESVT (underlined) andEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT CD86 mutantHQGLSSPVTKSFNRGECSGGGGSGGGGSAPL 1040 KIQAYFNETADLPCQFANSQNLSLSELVVFWQDQENLVLNEVYLGKERFDSVDSKYMGRTSFDSD SWTLRLHNLQIKDKGRYQCIIHHKKPTGMINIHQMNSELSVLA LIGHT CHAIN PREFERABLY ASSEMBLESWITH A HEAVY CHAIN COMPRISING THE 1170 VH SEQUENCETHUS, COMPLETE MOLECULE MAY BE DESIGNATED 1170/1263 128 1263 = 1171 ntGACATCCAGATGACCCAGTCTCCATCCTCCC light chain VL,TGAGCGCATCTGTAGGAGACCGCGTCACCAT with constantCACTTGCCGGGCAAGTCAGAGCATTAGCAGC kappa TATTTAAATTGGTATCAGCAGAAACCAGGGAAsequence, linker AGCCCCTAAGCTCCTGATCTATGCTGCATCC and CD86AGTTTGCAAAGTGGGGTCCCATCACGTTTCA mutant 1040GTGGCAGTGGAAGCGGGACAGATTTCACTCT CACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGGGTCATGGTTCT TACCCGCACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACgtgagtcgtacgctagcaagcttgatatcgaattctaaactctgagggggtcggatgacgtggccattctttgcctaaagcattgagtttactgcaaggtcagaaaagcatgcaaagccctcagaatggctgcaaagagctccaacaaaacaatttagaactttattaaggaatagggggaagctaggaagaaactcaaaacatcaagattttaaatacgcttcttggtctccttgctataattatctgggataagcatgctgttttctgtctgtccctaacatgccctgtgattatccgcaaacaacacacccaagggcagaactttgttacttaaacaccatcctgtttgcttctttcctcagGAACTGTGGCTGCACCATCTGTOTTCA TCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATA ACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAAC TCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCT GACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGG GCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTAGCGGAGGAGGAGGAAG CGGAGGAGGAGGAAGCGCCCCCCTCAAAATCCAAGCGTACTTCAACGAAACTGCAGACTTA CCGTGTCAGTTTGCCAATTCGCAGAATCTGAGCCTGAGCGAACTGGTGGTTTTCTGGCAGGA TCAGGAGAACCTGGTTCTGAACGAAGTCTATCTGGGCAAAGAGCGGTTCGACAGCGTGGAC AGCAAGTATATGGGCCGCACCAGCTTTGATAGCGACAGCTGGACCCTGCGTCTGCACAATCT GCAAATCAAAGATAAGGGTAGGTACCAGTGCATTATCCACCATAAGAAGCCGACGGGTATGA TTAATATTCACCAAATGAACTCCGAGTTGTCTGTCCTGGCG 129 1141 = 1135 aa DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNlight chain VL, WYQQKPGKAPKLLIYAASSLQSGVPSRFSGSG with constantSGTDFTLTISSLQPEDFATYYCQQSYSTPYTFG kappaQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVV sequence, linkerCLLNNFYPREAKVQWKVDNALQSGNSQESVTE (underlined) andQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH CD86 mutantQGLSSPVTKSFNRGECSGGGGSGGGGSAPLKI 1040 QAYFNETADLPCQFANSQNLSLSELVVFWQDQENLVLNEVYLGKERFDSVDSKYMGRTSFDSDS WTLRLHNLQIKDKGRYQCIIHHKKPTGMINIHQMNSELSVLA LIGHT CHAIN PREFERABLY ASSEMBLESWITH A HEAVY CHAIN COMPRISING ANY ONEOF THE 1164, 1168, 1520, OR 1542 VH SEQUENCESTHUS, COMPLETE MOLECULES MAY BEDESIGNATED 1164/1141, 1168/1141, 1520/1141 OR 1542/1141 130 1141 = 1135nt GACATCCAGATGACCCAGTCTCCATCCTCCC light chain VL,TGAGCGCATCTGTAGGAGACCGCGTCACCAT with constantCACTTGCCGGGCAAGTCAGAGCATTAGCAGC kappa TATTTAAATTGGTATCAGCAGAAACCAGGGAAsequence, linker AGCCCCTAAGCTCCTGATCTATGCTGCATCC and CD86AGTTTGCAAAGTGGGGTCCCATCACGTTTCA mutant 1040GTGGCAGTGGAAGCGGGACAGATTTCACTCT CACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGAGTTACAGTACC CCTTATACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACgtgagtcgtacgctagcaagcttgatatcgaattctaaactctgagggggtcggatgacgtggccattctttgcctaaagcattgagtttactgcaaggtcagaaaagcatgcaaagccctcagaatggctgcaaagagctccaacaaaacaatttagaactttattaaggaatagggggaagctaggaagaaactcaaaacatcaagattttaaatacgcttcttggtctccttgctataattatctgggataagcatgctgttttctgtctgtccctaacatgccctgtgattatccgcaaacaacacacccaagggcagaactttgttacttaaacaccatcctgtttgcttctttcctcagGAACTGTGGCTGCACCATCTGTCTTCATCT TCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACT TCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCC CAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGA CGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGC CTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTAGCGGAGGAGGAGGAAGCG GAGGAGGAGGAAGCGCCCCCCTCAAAATCCAAGCGTACTTCAACGAAACTGCAGACTTACC GTGTCAGTTTGCCAATTCGCAGAATCTGAGCCTGAGCGAACTGGTGGTTTTCTGGCAGGATC AGGAGAACCTGGTTCTGAACGAAGTCTATCTGGGCAAAGAGCGGTTCGACAGCGTGGACAG CAAGTATATGGGCCGCACCAGCTTTGATAGCGACAGCTGGACCCTGCGTCTGCACAATCTGC AAATCAAAGATAAGGGTAGGTACCAGTGCATTATCCACCATAAGAAGCCGACGGGTATGATTA ATATTCACCAAATGAACTCCGAGTTGTCTGTC CTGGCG131 1581 = 1515 aa DIQMTQSPSSLSASVGDRVTITCRASQSISSYLN light chain VL,WYQQKPGKAPKLLIYAASSLQSGVPSRFSGSG with constantSGTDFTLTISSLQPEDFATYYCQQGDYTLFTFG kappaQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVV sequence, linkerCLLNNFYPREAKVQWKVDNALQSGNSQESVTE (underlined) andQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTH CD86 mutantQGLSSPVTKSFNRGECSGGGGSGGGGSAPLKI 1040 QAYFNETADLPCQFANSQNLSLSELVVFWQDQENLVLNEVYLGKERFDSVDSKYMGRTSFDSDS WTLRLHNLQIKDKGRYQCIIHHKKPTGMINIHQMNSELSVLA LIGHT CHAIN PREFERABLY ASSEMBLESWITH A HEAVY CHAIN COMPRISING THE 1514 VH SEQUENCETHUS, COMPLETE MOLECULE MAY BE DESIGNATED 1514/1581 132 1581 = 1515 ntGACATCCAGATGACCCAGTCTCCATCCTCCC light chain VL,TGAGCGCATCTGTAGGAGACCGCGTCACCAT with constantCACTTGCCGGGCAAGTCAGAGCATTAGCAGC kappa TATTTAAATTGGTATCAGCAGAAACCAGGGAAsequence, linker AGCCCCTAAGCTCCTGATCTATGCTGCATCC and CD86AGTTTGCAAAGTGGGGTCCCATCACGTTTCA mutant 1040GTGGCAGTGGAAGCGGGACAGATTTCACTCT CACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGGGTGATTACACT CTGTTCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACgtgagtcgtacgctagcaagcttgatatcgaattctaaactctgagggggtcggatgacgtggccattctttgcctaaagcattgagtttactgcaaggtcagaaaagcatgcaaagccctcagaatggctgcaaagagctccaacaaaacaatttagaactttattaaggaatagggggaagctaggaagaaactcaaaacatcaagattttaaatacgcttcttggtctccttgctataattatctgggataagcatgctgttttctgtctgtccctaacatgccctgtgattatccgcaaacaacacacccaagggcagaactttgttacttaaacaccatcctgtttgcttctttcctcagGAACTGTGGCTGCACCATCTGTCTTCATCT TCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACT TCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCC CAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGA CGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGC CTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTAGCGGAGGAGGAGGAAGCG GAGGAGGAGGAAGCGCCCCCCTCAAAATCCAAGCGTACTTCAACGAAACTGCAGACTTACC GTGTCAGTTTGCCAATTCGCAGAATCTGAGCCTGAGCGAACTGGTGGTTTTCTGGCAGGATC AGGAGAACCTGGTTCTGAACGAAGTCTATCTGGGCAAAGAGCGGTTCGACAGCGTGGACAG CAAGTATATGGGCCGCACCAGCTTTGATAGCGACAGCTGGACCCTGCGTCTGCACAATCTGC AAATCAAAGATAAGGGTAGGTACCAGTGCATTATCCACCATAAGAAGCCGACGGGTATGATTA ATATTCACCAAATGAACTCCGAGTTGTCTGTC CTGGCG133 1585 = 1527 aa DIQMTQSPSSLSASVGDRVTITCRASQSISSYLN light chain VL,WYQQKPGKAPKLLIYAASSLQSGVPSRFSGSG with constantSGTDFTLTISSLQPEDFATYYCQQYGSDSLLTF kappaGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASV sequence, linkerVCLLNNFYPREAKVQWKVDNALQSGNSQESVT (underlined) andEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT CD86 mutantHQGLSSPVTKSFNRGECSGGGGSGGGGSAPL 1040 KIQAYFNETADLPCQFANSQNLSLSELVVFWQDQENLVLNEVYLGKERFDSVDSKYMGRTSFDSD SWTLRLHNLQIKDKGRYQCIIHHKKPTGMINIHQMNSELSVLA LIGHT CHAIN PREFERABLY ASSEMBLESWITH A HEAVY CHAIN COMPRISING THE 1526 VH SEQUENCETHUS, COMPLETE MOLECULE MAY BE DESIGNATED 1526/1585 134 1585 = 1527 ntGACATCCAGATGACCCAGTCTCCATCCTCCC light chain VL,TGAGCGCATCTGTAGGAGACCGCGTCACCAT with constantCACTTGCCGGGCAAGTCAGAGCATTAGCAGC kappa TATTTAAATTGGTATCAGCAGAAACCAGGGAAsequence, linker AGCCCCTAAGCTCCTGATCTATGCTGCATCC and CD86AGTTTGCAAAGTGGGGTCCCATCACGTTTCA mutant 1040GTGGCAGTGGAAGCGGGACAGATTTCACTCT CACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACGGTTCTGAT TCTCTGCTCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACgtgagtcgtacgctagcaagcttgatatcgaattctaaactctgagggggtcggatgacgtggccattctttgcctaaagcattgagtttactgcaaggtcagaaaagcatgcaaagccctcagaatggctgcaaagagctccaacaaaacaatttagaactttattaaggaatagggggaagctaggaagaaactcaaaacatcaagattttaaatacgcttcttggtctccttgctataattatctgggataagcatgctgttttctgtctgtccctaacatgccctgtgattatccgcaaacaacacacccaagggcagaactttgttacttaaacaccatcctgtttgcttctttcctcagGAACTGTGGCTGCACCATCTGTOTTCA TCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATA ACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAAC TCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCT GACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGG GCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTAGCGGAGGAGGAGGAAG CGGAGGAGGAGGAAGCGCCCCCCTCAAAATCCAAGCGTACTTCAACGAAACTGCAGACTTA CCGTGTCAGTTTGCCAATTCGCAGAATCTGAGCCTGAGCGAACTGGTGGTTTTCTGGCAGGA TCAGGAGAACCTGGTTCTGAACGAAGTCTATCTGGGCAAAGAGCGGTTCGACAGCGTGGAC AGCAAGTATATGGGCCGCACCAGCTTTGATAGCGACAGCTGGACCCTGCGTCTGCACAATCT GCAAATCAAAGATAAGGGTAGGTACCAGTGCATTATCCACCATAAGAAGCCGACGGGTATGA TTAATATTCACCAAATGAACTCCGAGTTGTCTGTCCTGGCG

TABLE E Exemplary polynucleotides encoding B2 - CTLA-4 SEQ ID 25 900CTCAAAATCCAAGCGTACTTCAACGAAACTGCAGACTTACCGTGTCAGTTTGCCAATTCGCAGAATCAAAGCCTGAGCGAACTGGTGGTTTTCTGGCAGGATCAGGAGAACCTGGTTCTGAACGAAGTCTATCTGGGCAAAGAGAAATTCGACAGCGTGGACAGCAAGTATATGGGCCGCACCAGCTTTGATAGCGACAGCTGGACCCTGCGTCTGCACAATCTGCAAATCAAAGATAAGGGTATCTACCAGTGCGTGATCCACCATAAGAAGCCGAGCGGTCTGGTGAAGATTCACGAGATGA ACTCCGAGTTGTCTGTCCTGGCG 26901 CTCAAAATCCAAGCGTACTTCAACGAAACTGCAGACTTACCGTGTCAGTTTGCCAATTCGCAGAATCTGACCCTGAGCGAACTGGTGGTTTTCTGGCAGGATCAGGAGAACCTGGTTCTGAACGAAGTCTATCTGGGCAAAGAGAAATTCGACAGCGTGCATAGCAAGTATATGGGCCGCACCAGCTTTGATAGCGACAGCTGGACCCTGCGTCTGCACAATCTGCAAATCAAAGATAAGGGTATCTACCAGTGCGTGATCCACCATAAGAAGCCGACGGGTATGATTAAGATTCACGAGATGAA CTCCGAGTTGTCTGTCCTGACC 27904 CTCAAAATCCAAGCGTACTTCAACGAAACTGCAGACTTACCGTGTCAGTTTGCCAATTCGCAGAATCAAAGCCTGAGCGAACTGATCGTTTTCTGGCAGGATCAGGAGAACCTGGTTCTGAACGAAGTCTATCTGGGCAAAGAGCGGTTCGACGCCGTGGACAGCAAGTATATGGGCCGCACCAGCTTTGATAGCGACAGCTGGACCCTGCGTCTGCACAATCTGCAAATCAAAGATAAGGGTATCTACCAGTGCATTATCCACCATAAGAAGCCGAGCGGTATGGTGAAGATTCACCAAATGGA CTCCGAGTTGTCTGTCCTGGCG 28906 CTCAAAATCCAAGCGTACATCAACGAAACTGCAGACTTACCGTGTCAGTTTGCCAATTCGCAGAATCTGAGCCTGAGCGAACTGGTGGTTTTCTGGCAGGATCAGGAGAACCTGGTTCTGAACGAAGTCTATCTGGGCAAAGAGCGGTTCGACAGCGTGGACAGCAAGTATATGGGCCGCACCAGCTTTGATAGCGACAGCTGGACCCTGCGTCTGCACAATCTGCAAATCAAAGATAAGGGTTTCTACCAGTGCATTATCCACCATAAGAAGCCGACGGGTCTGGTGAAGATTCACGAGATGA ACTCCGAGTTGTCTGTCCTGGCG 29907 CTCAAAATCCAAGCGTACTTCAACGAAACTGCAGACTTACCGTGTCAGTTTGCCAATTCGCAGAATCAAAGCCTGAGCGAACTGGTGGTTTTCTGGCAGGATCAGGAGAACCTGGTTCTGAACGAAGTCTATCTGGGCAAAGAGAAATTCGACAGCGTGCATAGCAAGTATATGGGCCGCACCAGCTTTGATAGCGACAGCTGGACCCTGCGTCTGCACAATCTGCAAATCAAAGATAAGGGTCTGTACCAGTGCATTATCCACCATAAGAAGCCGACGGGTATGATTAAGATTCACGAGATGAA CTCCGAGTTGTCTGTCCTGGCG 30908 CTCAAAATCCAAGCGTACTTCAACGAAACTGCAGACTTACCGTGTCAGTTTGCCAATTCGCAGAATCAAAGCCTGAGCGAACTGGTGGTTTTCTGGCAGGATCAGGAGAACCTGGTTCTGAACGAAGTCTATCTGGGCAAAGAGAAATTCGACAGCGTGCATAGCAAGTATATGGGCCGCACCAGCTTTGATAGCGACAGCTGGACCCTGCGTCTGCACAATCTGCAAATCAAAGATAAGGGTATCTACCAGTGCATTATCCACCATAAGAAGCCGACGGGTATGGTGAAGATTCACGAGATGA ACTCCGAGTTGTCTGTCCTGGCG 31910 CTCAAAATCCAAGCGTACTTCAACGAAACTGCAGACTTACCGTGTCAGTTTGCCAATTCGCAGAATCAAAGCCTGAGCGAACTGGTGGTTTTCTGGCAGGATCAGGAGAACCTGGTTCTGAACGAAGTCTATCTGGGCAAAGAGAAATTCGACAGCGTGGACAGCAAGTATATGGGCCGCACCAGCTTTGATAGCGACAGCTGGACCCTGCGTCTGCACAATCTGCAAATCAAAGATAAGGGTATCTACCAGTGCATTATCCACCATAAGAAGCCGACGGGTATGGTGAAGATTCACGAGATGA ACTCCGAGTTGTCTGTCCTGGCG 32915 CTCAAAATCCAAGCGTACTTCAACGAAACTGCAGACTTACCGTGTCAGTTTGCCAATTCGCAGAATCAAAGCCTGAGCGAACTGGTGGTTTTCTGGCAGGATCAGGAGAACCTGATCCTGAACGAAGTCTATCTGGGCAAAGAGAAATTCGACAGCGTGGACAGCAAGTATATGGGCCGCACCAGCTTTGATAGCGACAGCTGGACCCTGCGTCTGCACAATCTGCAAATCAAAGATAAGGGTTTCTACCAGTGCATTATCCACCATAAGAAGCCGAGCGGTCTGATTAAGATTCACCAAATGGA CTCCGAGTTGTCTGTCCTGGCG 33938 CTCAAAATCCAAGCGTACTTCAACGAAACTGCAGACTTACCGTGTCAGTTTGCCAATTCGCAGAATCTGAGCCTGAGCGAACTGGTGGTTTTCTGGCAGGATCAGGAGAACCTGATCCTGAACGAAGTCTATCTGGGCAAAGAGCGGTTCGACAGCGTGCATAGCAAGTATATGGGCCGCACCAGCTTTGATAGCGACAGCTGGACCCTGCGTCTGCACAATCTGCAAATCAAAGATAAGGGTCTGTACCAGTGCATTATCCACCATAAGAAGCCGAGCGGTATGGTGAAGATTCACGAGATGA ACTCCGAGTTGTCTGTCCTGGCG 341038 GCCCCCCTCAAAATCCAAGCGTACTTCAACGAAACTGCAGACTTACCGTGTCAGTTTGCCAATTCGCAGAATCTGAGCCTGAGCGAACTGGTGGTTTTCTGGCAGGATCAGGAGAACCTGGTTCTGAACGAAGTCTATCTGGGCAAAGAGAAATTCGACAGCGTGGACAGCAAGTATATGGGCCGCACCAGCTTTGATAGCGACAGCTGGACCCTGCGTCTGCACAATCTGCAAATCAAAGATAAGGGTATCTACCAGTGCATTATCCACCATAAGAAGCCGACGGGTATGGTGAAGATTCACGAGATGAACTCCGAGTTGTCTGTCCTGGCG 35 1039GCCCCCCTCAAAATCCAAGCGTACTTCAACGAAACTGCAGACTTACCGTGTCAGTTTGCCAATTCGCAGAATCTGAGCCTGAGCGAACTGGTGGTTTTCTGGCAGGATCAGGAGAACCTGGTTCTGAACGAAGTCTATCTGGGCAAAGAGAAATTCGACAGCGTGAGTAGCAAGTATATGGGCCGCACCAGCTTTGATAGCGACAGCTGGACCCTGCGTCTGCACAATCTGCAAATCAAAGATAAGGGTATCTACCAGTGCATTATCCACCATAAGAAGCCGAGCGGTATGGTGAAGATTCACCAAATGGACTCCGAGTTGTCTGTCCTGGCG 36 1040GCCCCCCTCAAAATCCAAGCGTACTTCAACGAAACTGCAGACTTACCGTGTCAGTTTGCCAATTCGCAGAATCTGAGCCTGAGCGAACTGGTGGTTTTCTGGCAGGATCAGGAGAACCTGGTTCTGAACGAAGTCTATCTGGGCAAAGAGCGGTTCGACAGCGTGGACAGCAAGTATATGGGCCGCACCAGCTTTGATAGCGACAGCTGGACCCTGCGTCTGCACAATCTGCAAATCAAAGATAAGGGTAGGTACCAGTGCATTATCCACCATAAGAAGCCGACGGGTATGATTAATATTCACCAAATGAACTCCGAGTTGTCTGTCCTGGCG 37 1041GCCCCCCTCAAAATCCAAGCGTACCTCAACGAAACTGCAGACTTACCGTGTCAGTTTGCCAATTCGCAGAATCTGAGCCTGAGCGAACTGGTGGTTTTCTGGCAGGATCAGGAGAACCTGGTTCTGAACGAAGTCTATCTGGGCAAAGAGAAATTCGACAGCGTGGACAGCAAGTATATGGGCCGCACCAGCTTTGATAGCGACAGCTGGACCCTGCGTCTGCACAATCTGCAAATCAAAGATAAGGGTATCTACCAGTGCATTATCCACCATAAGAAGCCGACGGGTCTGGTGAAGATTCACGAGATGAACTCCGAGTTGTCTGTCCTGGCG 38 1042GCCCCCCTCAAAATCCAAGCGTACTTCAACGAAACTGCAGACTTACCGTGTCAGTTTGCCAATTCGCAGAATCTGAGCCTGAGCGAACTGGTGGTTTTCTGGCAGGATCAGGAGAACCTGGTTCTGAACGAAGTCTATCTGGGCAAAGAGATTTTCGACAGCGTGAGTAGCAAGTATATGGGCCGCACCAGCTTTGATAGTGACAGCTGGACCCTGCGTCTGCACAATCTGCAAATCAAAGATAAGGGTATCTACCAGTGCATTATCCACCATAAGAAGCCGAGCGGTATGGTGAAGATTCACCAAATGGACTCCGAGTTGTCTGTCCTGGCG 39 1043GCCCCCCTCAAAATCCAAGCGTACTTCAACGAAACTGCAGACTTACCGTGTCAGTTTGCCAATTCGCAGAATCTGAGCCTGAGCGAACTGGTGGTTTTCTGGCAGGATCAGGAGAACCTGGTTCTGAACGAAGTCTATCTGGGCAAAGAGAAATTCGACAGCGTGGATAGCAAGTATATGGGCCGCACCAGCTTTGATAGCGACAGCTGGACCCTGCGTCTGCACAATCTGCAAATCAAAGATAAGGGTATCTACCAGTGCATTATCCACCATAAGAAGCCGACGGGTATGATTAAGATTCACGAGATGAACTCCGAGTTGTCTGTCCTGGCG 40 1044GCCCCCCTCAAAATCCAAGCGTACTTCAACGAAACTGCAGACTTACCGTGTCAGTTTGCCAATTCGCAGAATCTGACCCTGAGCGAACTGGTGGTTTTCTGGCAGGATCAGGAGAACCTGGTTCTGAACGAAGTCTATCTGGGCAAAGAGAAATTCGACAGCGTGTCTAGCAAGTATATGGGCCGCACCAGCTTTGATAGCGACAGCTGGACCCTGCGTCTGCACAATCTGCAAATCAAAGATAAGGGTATCTACCAGTGCATTATCCACCATAAGAAGCCGACGGGTATGATTAAGATTCACGAGATGAGCTCCGAGTTGTCTGTCCTGGCG 41 1045GCCCCCCTCAAAATCCAAGCGTACTTCAACGAAACTGCAGACTTACCGTGTCAGTTTGCCAATTCGCAGAATCTGACCCTGAGCGAACTGGTGGTTTTCTGGCAGGATCAGGAGAACCTGGTTCTGAACGAAGTCTATCTGGGCAAAGAGAAATTCGACAGCGTGGACAGCAAGTATATGGGCCGCACCAGCTTTGATAGCGACAGCTGGACCCTGCGTCTGCACAATCTGCAAATCAAAGATAAGGGTCTGTACCAGTGCATTATCCACCATAAGAAGCCGACGGGTCTGGTGAAGATTCACGAGATGAACTCCGAGTTGTCTGTCCTGGCG 42 1046GCCCCCCTCAAAATCCAAGCGTACTTCAACGAAACTGCAGACTTACCGTGTCAGTTTGCCAATTCGCAGAATCAAAGCCTGAGCGAACTGGTGGTTTTCTGGCAGGATCAGGAGAACCTGGTTCTGAACGAAGTCTATCTGGGCAAAGAGAAATTCGACAGCGTGGACAGCAAGTATATGGGCCGCACCAGCTTTGATAGCGACAGCTGGACCCTGCGTCTGCACAATCTGCAAATCGAAGATAAGGGTATCTACCAGTGCATTATCCACCATAAGAAGCCGAGCGGTATGGTGAAGATTCACCAAATGGACTCCGAGTTGTCTGTCCTGGCG 43 1047GCCCCCCTCAAAATCCAAGCGTACTTCAACGAAACTGCAGACTTACCGTGTCAGTTTGCCAATTCGCAGAATCTGAGCCTGAGCGAACTGGTGGTTTTCTGGCAGGATCAGGAGAACCTGGTTCTGAACGAAGTCTATCTGGGCAAAGAGAAATTCGACAGCGTGGACAGCAAGTATATGGGCCGCACCAGCTTTGATAGCGACAGCTGGACCCTGCGTCTGCACAATCTGCAAATCAAAGATAAGGGTATCTACCAGTGCATTATCCACCATAAGAAGCCGACGGGTCTGGTGAAGATTCACGAGATGAACTCCGAGTTGTCTGTCCTGGCG

TABLE F Exemplary sequences SEQ ID NO DESIGNATION SEQUENCE 149Fusion of: EVQLLESGGGLVQPGGSLRLSCAASGFTFYGSSMYW VH1: 1164 (boldVRQAPGKGLEWVSGIYSSGGYTSYADSVKGRFTISRD underlined)NSKNTLYLQMNSLRAEDTAVYYCARGVPHGYFDYWG IgG1 constant QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV domains (italics)KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS LinkerSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD (underlined)KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV VH2: 1204 (bold)TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ VL2: 1205 (bold,YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE italics)KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK GGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSL RLSCAASGFTFSSYYMGWVRQAPGKGLEWVSGIGSYYGYTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT AVYYCARAYYDYNYYYAYFDYWGQGTLVTVSSGGGG SGGGGSGGGGS

This sequence typically expressed together with a lightchain sequence comprising VL1 1135 (SEQ ID NO: 97)fused to kappa sequence (SEQ ID NO: 136) to assemblea 1164/1135-1204/1205 bispecific antibody. 150 NucleotideGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGT sequenceACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAG encoding SEQ IDCCAGCGGATTCACCTTTTACGGTTCTTCTATGTACTG NO: 149GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGG GTCTCAGGTATTTACTCTTCTGGTGGTTACACATCTTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCGGTGTTCCTCATGGTTACTTTGACTATT GGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGAGTTaaCgtacgctagcaagctttctggggcaggccaggcctgaccttggctttggggcagggagggggctaaggtgaggcaggtggcgccagccaggtgcacacccaatgcccatgagcccagacactggacgctgaacctcgcggacagttaagaacccaggggcctctgcgccctgggcccagctctgtcccacaccgcggtcacatggcaccacctctcttgcagCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGC ACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCC CTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGgtgagaggccagcacagggagggagggtgtctgctggaagccaggctcagcgctcctgcctggacgcatcccggctatgcagccccagtccagggcagcaaggcaggccccgtctgcctcttcacccggaggcctctgcccgccccactcatgctcagggagagggtcttctggctttttccccaggctctgggcaggcacaggctaggtgcccctaacccaggccctgcacacaaaggggcaggtgctgggctcagacctgccaagagccatatccgggaggaccctgcccctgacctaagcccaccccaaaggccaaactctccactccctcagctcggacaccttctctcctcccagattccagtaactcccaatcttctctctgcagAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGgtaagccagcccaggcctcgccctccagctcaaggcgggacaggtgccctagagtagcctgcatccagggacaggccccagccgggtgctgacacgtccacctccatctcttcctcagCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTC CCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGT ACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCG TGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGgtgggacccgtggggtgcgagggccacatggacagaggccggctcggcccaccctctgccctgagagtgaccgctgtaccaacctctgtccctacagGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAAC CAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGG GCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCA AGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCT CCGGGTAAAGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGAGGTGCAGCTGCTC GAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTTCTTACTACATGGGTTGGGTCCGCCAGGCTC CAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTGGTTCTTACTACGGTTACACAGGTTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCGCTTACTACGACTACAACTACTACTACGCTTACTTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGT GGAGGCGGTTCAGGCGGAGGTGGATCCGGCGGTGGCGGATCGGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGG TCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTCTGTTCCGCACTACCCGTTCACTTTTGGCCAGGGGACCAAGCTGG AGATCAAACGC 151 Fusion of:EVQLLESGGGLVQPGGSLRLSCAASGFTFGGYYMSW VH1: 1166 (boldVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRD underlined)NSKNTLYLQMNSLRAEDTAVYYCARYDYASMDYWG IgG1 constant QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV domains (italics)KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS LinkerSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD (underlined)KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV VH2: 1204 (bold)TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ VL2: 1205 (bold,YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE italics)KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK GGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSL RLSCAASGFTFSSYYMGWVRQAPGKGLEWVSGIGSYYGYTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT AVYYCARAYYDYNYYYAYFDYWGQGTLVTVSSGGGG SGGGGSGGGGS

This sequence typically expressed together with a lightchain sequence comprising VL1 1167 (SEQ ID NO: 89)fused to kappa sequence (SEQ ID NO: 136) to assemblea 1166/1167-1204/1205 bispecific antibody. 152 NucleotideGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGT sequenceACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAG encoding SEQ IDCCAGCGGATTCACCTTTGGTGGTTACTACATGTCTT NO: 151GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTG GGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTACGACTACGCTTCTATGGACTATTG GGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGAGTTaaCgtacgctagcaagctttctggggcaggccaggcctgaccttggctttggggcagggagggggctaaggtgaggcaggtggcgccagccaggtgcacacccaatgcccatgagcccagacactggacgctgaacctcgcggacagttaagaacccaggggcctctgcgccctgggcccagctctgtcccacaccgcggtcacatggcaccacctctcttgcagCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACC TCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGA ACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTC AGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGgtgagaggccagcacagggagggagggtgtctgctggaagccaggctcagcgctcctgcctggacgcatcccggctatgcagccccagtccagggcagcaaggcaggccccgtctgcctcttcacccggaggcctctgcccgccccactcatgctcagggagagggtcttctggctttttccccaggctctgggcaggcacaggctaggtgcccctaacccaggccctgcacacaaaggggcaggtgctgggctcagacctgccaagagccatatccgggaggaccctgcccctgacctaagcccaccccaaaggccaaactctccactccctcagctcggacaccttctctcctcccagattccagtaactcccaatcttctctctgcagAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGgtaagccagcccaggcctcgccctccagctcaaggcgggacaggtgccctagagtagcctgcatccagggacaggccccagccgggtgctgacacgtccacctccatctcttcctcagCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGT GGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTG GTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGgtgggacccgtggggtgcgagggccacatggacagaggccggctcggcccaccctctgccctgagagtgaccgctgtaccaacctctgtccctacagGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGG TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG GTAAAGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGAGGTGCAGCTGCTCGAGA GCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTTCTTACTACATGGGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTGGTTCTTACTACGGTTACACAGGTTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCGCTTACTACGACTACAACTACTACTACGCTTACTTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGG CGGTTCAGGCGGAGGTGGATCCGGCGGTGGCGGATCGGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTCTGTTCCGCACTACCCGTTCACTTTTGGCCAGGGGACCAAGCTGGAGATCAA ACGC 153 Fusion of:EVQLLESGGGLVQPGGSLRLSCAASGFTFSGSSMSW VH1: 1168 (boldVRQAPGKGLEWVSSISYYGGYTYYADSVKGRFTISRD underlined)NSKNTLYLQMNSLRAEDTAVYYCARYFPHYYFDYWG IgG1 constant QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV domains (italics)KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS LinkerSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD (underlined)KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV VH2: 1204 (bold)TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK GGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSL RLSCAASGFTFSSYYMGWVRQAPGKGLEWVSGIGSYYGYTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT AVYYCARAYYDYNYYYAYFDYWGQGTLVTVSSGGGG SGGGGSGGGGS

This sequence typically expressed together with a lightchain sequence comprising VL1 1135 (SEQ ID NO: 97)fused to kappa sequence (SEQ ID NO: 136) to assemblea 1168/1135-1204/1205 bispecific antibody. 154 NucleotideGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGT sequenceACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAG encoding SEQ IDCCAGCGGATTCACCTTTAGTGGTTCTTCTATGTCTTG NO: 153GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGG GTCTCATCTATTTCTTACTACGGTGGTTACACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTACTTCCCGCATTACTACTTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGAGTTaaCgtacgctagcaagctttctggggcaggccaggcctgaccttggctttggggcagggagggggctaaggtgaggcaggtggcgccagccaggtgcacacccaatgcccatgagcccagacactggacgctgaacctcgcggacagttaagaacccaggggcctctgcgccctgggcccagctctgtcccacaccgcggtcacatggcaccacctctcttgcagCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACC TCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGA ACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTC AGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGgtgagaggccagcacagggagggagggtgtctgctggaagccaggctcagcgctcctgcctggacgcatcccggctatgcagccccagtccagggcagcaaggcaggccccgtctgcctcttcacccggaggcctctgcccgccccactcatgctcagggagagggtcttctggctttttccccaggctctgggcaggcacaggctaggtgcccctaacccaggccctgcacacaaaggggcaggtgctgggctcagacctgccaagagccatatccgggaggaccctgcccctgacctaagcccaccccaaaggccaaactctccactccctcagctcggacaccttctctcctcccagattccagtaactcccaatcttctctctgcagAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGgtaagccagcccaggcctcgccctccagctcaaggcgggacaggtgccctagagtagcctgcatccagggacaggccccagccgggtgctgacacgtccacctccatctcttcctcagCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGT GGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTG GTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGgtgggacccgtggggtgcgagggccacatggacagaggccggctcggcccaccctctgccctgagagtgaccgctgtaccaacctctgtccctacagGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGG TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG GTAAAGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGAGGTGCAGCTGCTCGAGA GCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTTCTTACTACATGGGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTGGTTCTTACTACGGTTACACAGGTTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCGCTTACTACGACTACAACTACTACTACGCTTACTTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGG CGGTTCAGGCGGAGGTGGATCCGGCGGTGGCGGATCGGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTCTGTTCCGCACTACCCGTTCACTTTTGGCCAGGGGACCAAGCTGGAGATCAA ACGC 155 Fusion of:EVQLLESGGGLVQPGGSLRLSCAASGFTFGGYYMSW VH1: 1170 (boldVRQAPGKGLEWVSYIPGSGGSTYYADSVKGRFTISRD underlined)NSKNTLYLQMNSLRAEDTAVYYCARYDYYWMDYWG IgG1 constant QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV domains (italics)KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS LinkerSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD (underlined)KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV VH2: 1204 (bold)TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ VL2: 1205 (bold,YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE italics)KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK GGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSL RLSCAASGFTFSSYYMGWVRQAPGKGLEWVSGIGSYYGYTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT AVYYCARAYYDYNYYYAYFDYWGQGTLVTVSSGGGG SGGGGSGGGGS

This sequence typically expressed together with a lightchain sequence comprising VL1 1171 (SEQ ID NO: 93)fused to kappa sequence (SEQ ID NO: 136) to assemblea 1170/1171-1204/1205 bispecific antibody. 156 NucleotideGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGT sequenceACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAG encoding SEQ IDCCAGCGGATTCACCTTTGGTGGTTACTACATGTCTT NO: 155GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTG GGTCTCATACATTCCTGGTTCTGGTGGTTCTACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTACGACTACTACTGGATGGACTATTGG GGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGAGTTaaCgtacgctagcaagctttctggggcaggccaggcctgaccttggctttggggcagggagggggctaaggtgaggcaggtggcgccagccaggtgcacacccaatgcccatgagcccagacactggacgctgaacctcgcggacagttaagaacccaggggcctctgcgccctgggcccagctctgtcccacaccgcggtcacatggcaccacctctcttgcagCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACC TCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGA ACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTC AGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGgtgagaggccagcacagggagggagggtgtctgctggaagccaggctcagcgctcctgcctggacgcatcccggctatgcagccccagtccagggcagcaaggcaggccccgtctgcctcttcacccggaggcctctgcccgccccactcatgctcagggagagggtcttctggctttttccccaggctctgggcaggcacaggctaggtgcccctaacccaggccctgcacacaaaggggcaggtgctgggctcagacctgccaagagccatatccgggaggaccctgcccctgacctaagcccaccccaaaggccaaactctccactccctcagctcggacaccttctctcctcccagattccagtaactcccaatcttctctctgcagAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGgtaagccagcccaggcctcgccctccagctcaaggcgggacaggtgccctagagtagcctgcatccagggacaggccccagccgggtgctgacacgtccacctccatctcttcctcagCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGG ACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGT GGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTG GTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGgtgggacccgtggggtgcgagggccacatggacagaggccggctcggcccaccctctgccctgagagtgaccgctgtaccaacctctgtccctacagGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGG TCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG CCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGG GTAAAGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGAGGTGCAGCTGCTCGAGA GCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTTCTTACTACATGGGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTGGTTCTTACTACGGTTACACAGGTTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCGCTTACTACGACTACAACTACTACTACGCTTACTTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGG CGGTTCAGGCGGAGGTGGATCCGGCGGTGGCGGATCGGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTCTGTTCCGCACTACCCGTTCACTTTTGGCCAGGGGACCAAGCTGGAGATCAA ACGC 157 Fusion of:EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSW VH1: 1482 (boldVRQAPGKGLEWVSYISYYSGYTYYADSVKGRFTISRD underlined)NSKNTLYLQMNSLRAEDTAVYYCARGYGYLDYWGQ IgG1 constant GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVK domains (italics)DYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS LinkerVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDK (underlined)THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT VH2: 1204 (bold)CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY VL2: 1205 (bold,NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK italics)TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K GGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLR LSCAASGFTFSSYYMGWVRQAPGKGLEWVSGIGSYYGYTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA VYYCARAYYDYNYYYAYFDYWGQGTLVTVSSGGGGS GGGGSGGGGS

This sequence typically expressed together with a lightchain sequence comprising VL1 1483 (SEQ ID NO: 103)fused to kappa sequence (SEQ ID NO: 136) to assemblea 1482/1483-1204/1205 bispecific antibody. 158 NucleotideGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGT sequenceACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAG encoding SEQ IDCCAGCGGATTCACCTTTAGCAGCTATGCCATGAGCT NO: 157GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTG GGTCTCATACATTTCTTACTACTCTGGTTACACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCGGTTACGGTTACTTGGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGAGTTaaCgtacgctagcaagctttctggggcaggccaggcctgaccttggctttggggcagggagggggctaaggtgaggcaggtggcgccagccaggtgcacacccaatgcccatgagcccagacactggacgctgaacctcgcggacagttaagaacccaggggcctctgcgccctgggcccagctctgtcccacaccgcggtcacatggcaccacctctcttgcagCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCT GGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACT CAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGC AGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGgtgagaggccagcacagggagggagggtgtctgctggaagccaggctcagcgctcctgcctggacgcatcccggctatgcagccccagtccagggcagcaaggcaggccccgtctgcctcttcacccggaggcctctgcccgccccactcatgctcagggagagggtcttctggctttttccccaggctctgggcaggcacaggctaggtgcccctaacccaggccctgcacacaaaggggcaggtgctgggctcagacctgccaagagccatatccgggaggaccctgcccctgacctaagcccaccccaaaggccaaactctccactccctcagctcggacaccttctctcctcccagattccagtaactcccaatcttctctctgcagAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGgtaagccagcccaggcctcgccctccagctcaaggcgggacaggtgccctagagtagcctgcatccagggacaggccccagccgggtgctgacacgtccacctccatctcttcctcagCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCC CTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGAC GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCA GCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGgtgggacccgtggggtgcgagggccacatggacagaggccggctcggcccaccctctgccctgagagtgaccgctgtaccaacctctgtccctacagGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCC CCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGA CATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCG TGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAA GGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGAGGTGCAGCTGCTCGAGAGCGG GGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTTCTTACTACATGGGTTGGGTCCGCCAGGCTCCAGGGAAGG GGCTGGAGTGGGTCTCAGGTATTGGTTCTTACTACGGTTACACAGGTTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGT ATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCGCTTACTACGACTACAACTACTACTACGCTTACTTTGACTATTGGGGCCAGG GAACCCTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGATCCGGCGGTGGCGGATCGG ACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTCTGTTCCGCACTACCCGTTCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGC 159 Fusion of:EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSW VH1: 1520 (boldVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRD underlined)NSKNTLYLQMNSLRAEDTAVYYCARYYYSHGYYVYG IgG1 constant TLDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGT domains (italics)AALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS LinkerSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKK (underlined)VEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLM VH2: 1204 (bold)ISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKT VL2: 1205 (bold,KPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK italics)ALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK GGGGSGGGGSGGGGSEVQLLESGGGLV QPGGSLRLSCAASGFTFSSYYMGWVRQAPGKGLEWVSGIGSYYGYTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAYYDYNYYYAYFDYWGQGTLVT VSS GGGGSGGGGSGGGGS

This sequence typically expressed together with a lightchain sequence comprising VL1 1135 (SEQ ID NO: 97)fused to kappa sequence (SEQ ID NO: 136) to assemblea 1520/1135-1204/1205 bispecific antibody. 160 NucleotideGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGT sequenceACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAG encoding SEQ IDCCAGCGGATTCACCTTTAGCAGCTATGCCATGAGCT NO: 159GGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTG GGTCTCAGCTATTAGTGGTAGTGGTGGTAGCACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTACTACTACTCTCATGGTTACTACGTTTACGGTACTTTGGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGAGTTaaCgtacgctagcaagctttctggggcaggccaggcctgaccttggctttggggcagggagggggctaaggtgaggcaggtggcgccagccaggtgcacacccaatgcccatgagcccagacactggacgctgaacctcgcggacagttaagaacccaggggcctctgcgccctgggcccagctctgtcccacaccgcggtcacatggcaccacctctcttgcagCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGG CACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGA ACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAG TCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGgtgagaggccagcacagggagggagggtgtctgctggaagccaggctcagcgctcctgcctggacgcatcccggctatgcagccccagtccagggcagcaaggcaggccccgtctgcctcttcacccggaggcctctgcccgccccactcatgctcagggagagggtcttctggctttttccccaggctctgggcaggcacaggctaggtgcccctaacccaggccctgcacacaaaggggcaggtgctgggctcagacctgccaagagccatatccgggaggaccctgcccctgacctaagcccaccccaaaggccaaactctccactccctcagctcggacaccttctctcctcccagattccagtaactcccaatcttctctctgcagAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGgtaagccagcccaggcctcgccctccagctcaaggcgggacaggtgccctagagtagcctgcatccagggacaggccccagccgggtgctgacacgtccacctccatctcttcctcagCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACA TGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGG TGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACC GTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGgtgggacccgtggggtgcgagggccacatggacagaggccggctcggcccaccctctgccctgagagtgaccgctgtaccaacctctgtccctacagGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGG ATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGG AGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTC CTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCG TGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAGGAGGAGGA GGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGAGGTGCAGCTGCTCGAGAGCGGGGGAGGCTT GGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTTCTTACTACATGGGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGT GGGTCTCAGGTATTGGTTCTTACTACGGTTACACAGGTTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCGCTTACTACGACTACAACTACTACTACGCTTACTTTGACTATTGGGGCCAGGGAACCCTGGT CACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGATCCGGCGGTGGCGGATCGGACATCCAGATG ACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTCTGTTCCGCACTACCCGTTCACTTTTGGCC AGGGGACCAAGCTGGAGATCAAACGC 161Fusion of: EVQLLESGGGLVQPGGSLRLSCAASGFTFSGYSMYW VH1: 1526 (boldVRQAPGKGLEWVSGIGYSGYGTYYADSVKGRFTISRD underlined)NSKNTLYLQMNSLRAEDTAVYYCARYYFHDYAAYSL IgG1 constant DYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAA domains (italics)LGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LinkerLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE (underlined)PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMIS VH2: 1204 (bold)RTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP VL2: 1205 (bold,REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL italics)PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK GGGGSGGGGSGGGGSEVQLLESGGGLVQP GGSLRLSCAASGFTFSSYYMGWVRQAPGKGLEWVSGIGSYYGYTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAYYDYNYYYAYFDYWGQGTLVTVS S GGGGSGGGGSGGGGS

This sequence typically expressed together with a lightchain sequence comprising VL1 1527 (SEQ ID NO: 119)fused to kappa sequence (SEQ ID NO: 136) to assemblea 1526/1527-1204/1205 bispecific antibody. 162 NucleotideGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGT sequenceACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAG encoding SEQ IDCCAGCGGATTCACCTTTTCTGGTTACTCTATGTACTG NO: 161GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGG GTCTCAGGTATTGGTTACTCTGGTTACGGTACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTACTACTTCCATGACTACGCTGCTTACT CTTTGGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA GGTGAGTTaaCgtacgctagcaagctttctggggcaggccaggcctgaccttggctttggggcagggagggggctaaggtgaggcaggtggcgccagccaggtgcacacccaatgcccatgagcccagacactggacgctgaacctcgcggacagttaagaacccaggggcctctgcgccctgggcccagctctgtcccacaccgcggtcacatggcaccacctctcttgcagCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAG AGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGT CGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTAC TCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGgtgagaggccagcacagggagggagggtgtctgctggaagccaggctcagcgctcctgcctggacgcatcccggctatgcagccccagtccagggcagcaaggcaggccccgtctgcctcttcacccggaggcctctgcccgccccactcatgctcagggagagggtcttctggctttttccccaggctctgggcaggcacaggctaggtgcccctaacccaggccctgcacacaaaggggcaggtgctgggctcagacctgccaagagccatatccgggaggaccctgcccctgacctaagcccaccccaaaggccaaactctccactccctcagctcggacaccttctctcctcccagattccagtaactcccaatcttctctctgcagAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGgtaagccagcccaggcctcgccctccagctcaaggcgggacaggtgccctagagtagcctgcatccagggacaggccccagccgggtgctgacacgtccacctccatctcttcctcagCACCTGAACTCCTGGGGGGACCGTCAGTOTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGAT CTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACT GGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTA CCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGgtgggacccgtggggtgcgagggccacatggacagaggccggctcggcccaccctctgccctgagagtgaccgctgtaccaacctctgtccctacagGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAG AACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAA TGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACA GCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTG TCTCCGGGTAAAGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGAGGTGCAGCTG CTCGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTC ACCTTTTCTTCTTACTACATGGGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTGGTTCTTACTACGGTTACACAGGTTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCGCTTACTACGACTACAACTACTACTACGCTTACTTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA GGTGGAGGCGGTTCAGGCGGAGGTGGATCCGGCGGTGGCGGATCGGACATCCAGATGACCCAGTCTCCAT CCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGT GGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTCTGTTCCGCACTACCCGTTCACTTTTGGCCAGGGGACCAAG CTGGAGATCAAACGC 163 Fusion of:EVQLLESGGGLVQPGGSLRLSCAASGFTFGSSSMYW VH1: 1542 (boldVRQAPGKGLEWVSGIGYYSYSTSYADSVKGRFTISRD underlined)NSKNTLYLQMNSLRAEDTAVYYCARGYPHHYFDYWG IgG1 constant QGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV domains (italics)KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS LinkerSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCD (underlined)KTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV VH2: 1204 (bold)TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ VL2: 1205 (bold,YNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE italics)KTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK GGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSL RLSCAASGFTFSSYYMGWVRQAPGKGLEWVSGIGSYYGYTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDT AVYYCARAYYDYNYYYAYFDYWGQGTLVTVSSGGGG SGGGGSGGGGS

This sequence typically expressed together with a lightchain sequence comprising VL1 1135 (SEQ ID NO: 97)fused to kappa sequence (SEQ ID NO: 136) to assemblea 1542/1135-1204/1205 bispecific antibody. 164 NucleotideGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGT sequenceACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAG encoding SEQ IDCCAGCGGATTCACCTTTGGTTCTTCTTCTATGTACTG NO: 163GGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGG GTCTCAGGTATTGGTTACTACTCTTACTCTACATCTTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCGGTTACCCGCATCATTACTTTGACTATT GGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGAGTTaaCgtacgctagcaagctttctggggcaggccaggcctgaccttggctttggggcagggagggggctaaggtgaggcaggtggcgccagccaggtgcacacccaatgcccatgagcccagacactggacgctgaacctcgcggacagttaagaacccaggggcctctgcgccctgggcccagctctgtcccacaccgcggtcacatggcaccacctctcttgcagCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGC ACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGT GGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCC CTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGgtgagaggccagcacagggagggagggtgtctgctggaagccaggctcagcgctcctgcctggacgcatcccggctatgcagccccagtccagggcagcaaggcaggccccgtctgcctcttcacccggaggcctctgcccgccccactcatgctcagggagagggtcttctggctttttccccaggctctgggcaggcacaggctaggtgcccctaacccaggccctgcacacaaaggggcaggtgctgggctcagacctgccaagagccatatccgggaggaccctgcccctgacctaagcccaccccaaaggccaaactctccactccctcagctcggacaccttctctcctcccagattccagtaactcccaatcttctctctgcagAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGgtaagccagcccaggcctcgccctccagctcaaggcgggacaggtgccctagagtagcctgcatccagggacaggccccagccgggtgctgacacgtccacctccatctcttcctcagCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTC CCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGT ACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCG TGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGgtgggacccgtggggtgcgagggccacatggacagaggccggctcggcccaccctctgccctgagagtgaccgctgtaccaacctctgtccctacagGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAAC CAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGG GCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCA AGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCT CCGGGTAAAGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGAGGTGCAGCTGCTC GAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTTCTTACTACATGGGTTGGGTCCGCCAGGCTC CAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTGGTTCTTACTACGGTTACACAGGTTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCGCTTACTACGACTACAACTACTACTACGCTTACTTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGT GGAGGCGGTTCAGGCGGAGGTGGATCCGGCGGTGGCGGATCGGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGG TCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTCTGTTCCGCACTACCCGTTCACTTTTGGCCAGGGGACCAAGCTGG AGATCAAACGC

TABLE G Exemplary sequences SEQ ID NO. DESIGNATION SEQUENCE 165Fusion of: DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQ VL1: 1135 (boldQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLT underlined)ISSLQPEDFATYYCQQSYSTPYTFGQGTKLEIK RTVA kappa constantAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW domains (italics)KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD (i.e. fusion ofYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NOS: 97May assemble with any one of SEQ ID  and 136)NOs: 149, 153, 159 or 163 to form a  1164/1135-1204/1205, 1168/1135- 1204/1205, 1520/1135-1204/1205 or  1542/1135-1204/1205 bispecific antibody. 166 Nucleotide GACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGencoding SEQ ID CATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGC NO: 165AAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGAGTTACAGTACCCCTTATACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACgtgagtcgtacgctagcaagcttgatatcgaattctaaactctgagggggtcggatgacgtggccattctttgcctaaagcattgagtttactgcaaggtcagaaaagcatgcaaagccctcagaatggctgcaaagagctccaacaaaacaatttagaactttattaaggaatagggggaagctaggaagaaactcaaaacatcaagattttaaatacgcttcttggtctccttgctataattatctgggataagcatgctgttttctgtctgtccctaacatgccctgtgattatccgcaaacaacacacccaagggcagaactttgttacttaaacaccatcctgtttgcttctttcctcagGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGG GAGAGTGT 167 Fusion of:DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQ VL1: 1167 (boldQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLT underlined)ISSLQPEDFATYYCQQYYWYGLSTFGQGTKLEIK RTV kappa constantAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ domains (italics)WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA (i.e. fusion ofDYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NOS: 89May assemble with SEQ ID NO: 151 to  and 136)form a 1166/1167-1204/1205 bispecific antibody. 168 NucleotideGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCG encoding SEQ IDCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGC NO: 167AAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACTACTGGTACGGTCTGTCCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGTGAGTCgtacgctagcaagcttgatatcgaattctaaactctgagggggtcggatgacgtggccattctttgcctaaagcattgagtttactgcaaggtcagaaaagcatgcaaagccctcagaatggctgcaaagagctccaacaaaacaatttagaactttattaaggaatagggggaagctaggaagaaactcaaaacatcaagattttaaatacgcttcttggtctccttgctataattatctgggataagcatgctgttttctgtctgtccctaacatgccctgtgattatccgcaaacaacacacccaagggcagaactttgttacttaaacaccatcctgtttgcttctttcctcagGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACA GGGGAGAGTGT 169 Fusion of:DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQ VL1: 1171 (boldQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLT underlined)ISSLQPEDFATYYCQQGHGSYPHTFGQGTKLEIK RTV kappa constantAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ domains (italics) WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA (i.e. fusion ofDYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NOS: 93May assemble with SEQ ID NO: 155 to  and 136)form a 1170/1171-1204/1205 bispecific antibody 170 NucleotideGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCG encoding SEQ IDCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGC NO: 169AAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGGGTCATGGTTCTTACCCGCACACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGTGAGTCgtacgctagcaagcttgatatcgaattctaaactctgagggggtcggatgacgtggccattctttgcctaaagcattgagtttactgcaaggtcagaaaagcatgcaaagccctcagaatggctgcaaagagctccaacaaaacaatttagaactttattaaggaatagggggaagctaggaagaaactcaaaacatcaagattttaaatacgcttcttggtctccttgctataattatctgggataagcatgctgttttctgtctgtccctaacatgccctgtgattatccgcaaacaacacacccaagggcagaactttgttacttaaacaccatcctgtttgcttctttcctcagGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACA GGGGAGAGTGT 171 Fusion of:DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQ VL1: 1483 (boldQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLT underlined)ISSLQPEDFATYYCQQYGSLLTFGQGTKLEIK RTVAA kappa constantPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK domains (italics) VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY (i.e. fusion ofEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NOS:May assemble with SEQ ID NO: 157 to  103 and 136)form a 1482/1483-1204/1205 bispecific antibody 172 NucleotideGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCG encoding SEQ IDCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGC NO: 171AAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACGGTTCTCTGCTCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGTGAGTCgtacgctagcaagcttgatatcgaattctaaactctgagggggtcggatgacgtggccattctttgcctaaagcattgagtttactgcaaggtcagaaaagcatgcaaagccctcagaatggctgcaaagagctccaacaaaacaatttagaactttattaaggaatagggggaagctaggaagaaactcaaaacatcaagattttaaatacgcttcttggtctccttgctataattatctgggataagcatgctgttttctgtctgtccctaacatgccctgtgattatccgcaaacaacacacccaagggcagaactttgttacttaaacaccatcctgtttgcttctttcctcagGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAG AGTGT 173 Fusion of:DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQ VL1: 1527 (boldQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLT underlined)ISSLQPEDFATYYCQQYGSDSLLTFGQGTKLEIK RTV kappa constantAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ domains (italics) WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA (i.e. fusion ofDYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID NOS:May assemble with SEQ ID NO: 161 to  119 and 136)form 1526/1527-1204/1205 bispecific  antibody 174 NucleotideGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCG encoding SEQ IDCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGC NO: 173AAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACGGTTCTGATTCTCTGCTCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAACGTGAGTCgtacgctagcaagcttgatatcgaattctaaactctgagggggtcggatgacgtggccattctttgcctaaagcattgagtttactgcaaggtcagaaaagcatgcaaagccctcagaatggctgcaaagagctccaacaaaacaatttagaactttattaaggaatagggggaagctaggaagaaactcaaaacatcaagattttaaatacgcttcttggtctccttgctataattatctgggataagcatgctgttttctgtctgtccctaacatgccctgtgattatccgcaaacaacacacccaagggcagaactttgttacttaaacaccatcctgtttgcttctttcctcagGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACA GGGGAGAGTGT

TABLE H Exemplary CD137 binding domains and bispecific polypeptides for CD137 and CTLA-4 SEQ ID NO. CHAIN NO. TYPESEQUENCE 177 1205, light  aa DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKchain VL PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSVPHYPFTFGQGTKLEIK 178 1205, light  ntGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCA chain VLTCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTCTGTTCCGCACTACCCGTTCACTTTTGGCCAGGGGACCAAG CTGGAGATCAAA 179 1204, heavy aaEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYYMGWVRQ chain VHAPGKGLEWVSGIGSYYGYTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARAYYDYNYYYAYFDYWGQGT LVTVSS 180 1204, heavy ntGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAG chain VHCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTTCTTACTACATGGGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTGGTTCTTACTACGGTTACACAGGTTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCGCTTACTACGACTACAACTACTACTACGCTTACTTTGACTATTGGGGCCAGGGAACC CTGGTCACCGTCTCCTCA 181 1214 (VH)aa EVQLLESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSSIGSGGGYTGYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARVGHPFDYWGQGTLVTVSS 182 1214 (VH) ntGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTGGTTCTGGTGGTGGTTACACAGGTTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCGTTGGTCATCCGTTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 183 1215 (VL) aaDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQQDAYPHTFGQGTKLEIK 1841215 (VL) nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGGACGCTTACCCGCACACTTTTGGCCAGGGGACCAAGCTGGAG ATCAAA 185 1618 (VH) aaEVQLLESGGGLVQPGGSLRLSCAASGFTFSYGSMYWVRQAPGKGLEWVSSISSGSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSSYYGSYYSIDYWGQGTLV TVSS 186 1618 (VH) ntGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTTACGGTTCTATGTACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTTCTTCTGGTTCTGGTTCTACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTCTTCTTACTACGGTTCTTACTACTCTATTGACTATTGGGGCCAGGGAACCCTGGTC ACCGTCTCCTCA 187 1619 (VL) aaDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQQYYDNLPTFGQGTKLEIK188 1619 (VL) nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTACTACGACAACCTGCCCACTTTTGGCCAGGGGACCAAGCTG GAGATCAAA 189 1620 (VH) aaEVQLLESGGGLVQPGGSLRLSCAASGFTFSGYYMYWVRQAPGKGLEWVSGISSSGSYTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSVGPYFDYWGQGTLVTVSS 190 1620 (VH) ntGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTTCTGGTTACTACATGTACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTTCTTCTTCTGGTTCTTACACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTCTGTTGGTCCGTACTTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 191 1621 (VL) aaDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQQGVGPYTFGQGTKLEIK 1921621 (VL) nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGGGTGTTGGTCCGTACACTTTTGGCCAGGGGACCAAGCTGGAG ATCAAA 193 1626 (VH) aaEVQLLESGGGLVQPGGSLRLSCAASGFTFGGYSMYWVRQAPGKGLEWVSSIGGYYYSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARSYYGSIDYWGQGTLVTVSS 194 1626 (VH) ntGAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCCAGCGGATTCACCTTTGGTGGTTACTCTATGTACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTGGTGGTTACTACTACTCTACATACTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCTCTTACTACGGTTCTATTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA 195 1627 (VL) aaDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL QPEDFATYYCQQGTGYGPLTFGQGTKLEIK196 1627 (VL) nt GACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGGGTACTGGTTACGGTCCGCTCACTTTTGGCCAGGGGACCAAG CTGGAGATCAAA 197 1761 = 1205 ntGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCA Light chainTCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGT VL, withCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAA constant CCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCC kappa AGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGT sequence, GGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTG linker andCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTCT CD86 mutantGTTCCGCACTACCCGTTCACTTTTGGCCAGGGGACCAAG 1040 CTGGAGATCAAACGTgagtcgtacgctagcaagcttgat inclusive atcgaattctaaactctgagggggtcggatgacgtggcc intronattctttgcctaaagcattgagtttactgcaaggtcaga sequenceaaagcatgcaaagccctcagaatggctgcaaagagctccaacaaaacaatttagaactttattaaggaatagggggaagctaggaagaaactcaaaacatcaagattttaaatacgcttcttggtctccttgctataattatctgggataagcatgctgttttctgtctgtccctaacatgccctgtgattatccgcaaacaacacacccaagggcagaactttgttacttaaacaccatcctgtttgcttctttcctcagGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGCCCCCCTCAAAATCCAAGCGTACTTCAACGAAACTGCAGACTTACCGTGTCAGTTTGCCAATTCGCAGAATCTGAGCCTGAGCGAACTGGTGGTTTTCTGGCAGGATCAGGAGAACCTGGTTCTGAACGAAGTCTATCTGGGCAAAGAGCGGTTCGACAGCGTGGACAGCAAGTATATGGGCCGCACCAGCTTTGATAGCGACAGCTGGACCCTGCGTCTGCACAATCTGCAAATCAAAGATAAGGGTAGGTACCAGTGCATTATCCACCATAAGAAGCCGACGGGTATGATTAATATTCACCAAATGAACTCCGAGTTGT CTGTCCTGGCG 198 1761 = 1205 aaDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQK light chain PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL VL, with QPEDFATYYCQQSVPHYPFTFGQGTKLEIKRTVAAPSVF constantIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ kappaSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC sequence, EVTHQGLSSPVTKSFNRGECSGGGGSGGGGSAPLKIQAY linkerFNETADLPCQFANSQNLSLSELVVFWQDQENLVLNEVYL (underlined) GKERFDSVDSKYMGRTSFDSDSWTLRLHNLQIKDKGRYQ and CD86 CIIHHKKPTGMINIHQMNSELSVLA mutant 1040LIGHT CHAIN PREFERABLY ASSEMBLES WITH AHEAVY CHAIN COMPRISING THE 1204 VH  SEQUENCETHUS, COMPLETE MOLECULE MAY BE  DESIGNATED 1204/1761 199 1763 = 1215 ntGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCA Light chain TCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGT VL, with CAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAA constantCCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCC kappaAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGT sequence, GGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTG linkerCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGGAC and CD86GCTTACCCGCACACTTTTGGCCAGGGGACCAAGCTGGAG mutant 1040ATCAAACGTgagtcgtacgctagcaagcttgatatcgaattctaaactctgagggggtcggatgacgtggccattctttgcctaaagcattgagtttactgcaaggtcagaaaagcatgcaaagccctcagaatggctgcaaagagctccaacaaaacaatttagaactttattaaggaatagggggaagctaggaagaaactcaaaacatcaagattttaaatacgcttcttggtctccttgctataattatctgggataagcatgctgttttctgtctgtccctaacatgccctgtgattatccgcaaacaacacacccaagggcagaactttgttacttaaacaccatcctgtttgcttctttcctcagGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGCCCCCCTCAAAATCCAAGCGTACTTCAACGAAACTGCAGACTTACCGTGTCAGTTTGCCAATTCGCAGAATCTGAGCCTGAGCGAACTGGTGGTTTTCTGGCAGGATCAGGAGAACCTGGTTCTGAACGAAGTCTATCTGGGCAAAGAGCGGTTCGACAGCGTGGACAGCAAGTATATGGGCCGCACCAGCTTTGATAGCGACAGCTGGACCCTGCGTCTGCACAATCTGCAAATCAAAGATAAGGGTAGGTACCAGTGCATTATCCACCATAAGAAGCCGACGGGTATGATTAATATTCACCAAATGAACTCCGAGTTGTCTGTCC TGGCG 200 1763 = 1215 aaDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQK Light chain PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL VL, with QPEDFATYYCQQDAYPHTFGQGTKLEIKRTVAAPSVFIF constantPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG kappaNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV sequence, THQGLSSPVTKSFNRGECSGGGGSGGGGSAPLKIQAYFN linkerETADLPCQFANSQNLSLSELVVFWQDQENLVLNEVYLGK (underlined) ERFDSVDSKYMGRTSFDSDSWTLRLHNLQIKDKGRYQCI and CD86 IHHKKPTGMINIHQMNSELSVLA mutant 1040LIGHT CHAIN PREFERABLY ASSEMBLES WITH AHEAVY CHAIN COMPRISING THE 1214 VH  SEQUENCETHUS, COMPLETE MOLECULE MAY BE  DESIGNATED 1214/1763 201 1765 = 1619 ntGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCA Light chain TCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGT VL, with CAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAA constantCCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCC kappaAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGT sequence, GGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTG linkerCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTAC and CD86TACGACAACCTGCCCACTTTTGGCCAGGGGACCAAGCTG mutant 1040GAGATCAAACGTgagtcgtacgctagcaagcttgatatcgaattctaaactctgagggggtcggatgacgtggccattctttgcctaaagcattgagtttactgcaaggtcagaaaagcatgcaaagccctcagaatggctgcaaagagctccaacaaaacaatttagaactttattaaggaatagggggaagctaggaagaaactcaaaacatcaagattttaaatacgcttcttggtctccttgctataattatctgggataagcatgctgttttctgtctgtccctaacatgccctgtgattatccgcaaacaacacacccaagggcagaactttgttacttaaacaccatcctgtttgcttctttcctcagGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGCCCCCCTCAAAATCCAAGCGTACTTCAACGAAACTGCAGACTTACCGTGTCAGTTTGCCAATTCGCAGAATCTGAGCCTGAGCGAACTGGTGGTTTTCTGGCAGGATCAGGAGAACCTGGTTCTGAACGAAGTCTATCTGGGCAAAGAGCGGTTCGACAGCGTGGACAGCAAGTATATGGGCCGCACCAGCTTTGATAGCGACAGCTGGACCCTGCGTCTGCACAATCTGCAAATCAAAGATAAGGGTAGGTACCAGTGCATTATCCACCATAAGAAGCCGACGGGTATGATTAATATTCACCAAATGAACTCCGAGTTGTCTG TCCTGGCG 202 1765 = 1619 aaDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQK Light chain PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL VL, with QPEDFATYYCQQYYDNLPTFGQGTKLEIKRTVAAPSVFI constantFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS kappaGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE sequence, VTHQGLSSPVTKSFNRGECSGGGGSGGGGSAPLKIQAYF linkerNETADLPCQFANSQNLSLSELVVFWQDQENLVLNEVYLG (underlined) KERFDSVDSKYMGRTSFDSDSWTLRLHNLQIKDKGRYQC and CD86 IIHHKKPTGMINIHQMNSELSVLA mutant 1040LIGHT CHAIN PREFERABLY ASSEMBLES WITH AHEAVY CHAIN COMPRISING THE 1618 VH  SEQUENCETHUS, COMPLETE MOLECULE MAY BE  DESIGNATED 1618/1765 203 1767 = 1621 ntGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCA Light chain TCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGT VL, with CAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAA constantCCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCC kappaAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGT sequence, GGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTG linkerCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGGGT and CD86GTTGGTCCGTACACTTTTGGCCAGGGGACCAAGCTGGAG mutant 1040ATCAAACGTgagtcgtacgctagcaagcttgatatcgaattctaaactctgagggggtcggatgacgtggccattctttgcctaaagcattgagtttactgcaaggtcagaaaagcatgcaaagccctcagaatggctgcaaagagctccaacaaaacaatttagaactttattaaggaatagggggaagctaggaagaaactcaaaacatcaagattttaaatacgcttcttggtctccttgctataattatctgggataagcatgctgttttctgtctgtccctaacatgccctgtgattatccgcaaacaacacacccaagggcagaactttgttacttaaacaccatcctgtttgcttctttcctcagGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGCCCCCCTCAAAATCCAAGCGTACTTCAACGAAACTGCAGACTTACCGTGTCAGTTTGCCAATTCGCAGAATCTGAGCCTGAGCGAACTGGTGGTTTTCTGGCAGGATCAGGAGAACCTGGTTCTGAACGAAGTCTATCTGGGCAAAGAGCGGTTCGACAGCGTGGACAGCAAGTATATGGGCCGCACCAGCTTTGATAGCGACAGCTGGACCCTGCGTCTGCACAATCTGCAAATCAAAGATAAGGGTAGGTACCAGTGCATTATCCACCATAAGAAGCCGACGGGTATGATTAATATTCACCAAATGAACTCCGAGTTGTCTGTCC TGGCG 204 1767 = 1621 aaDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQK Light chain PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL VL, with QPEDFATYYCQQGVGPYTFGQGTKLEIKRTVAAPSVFIF constantPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSG kappaNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV sequence, THQGLSSPVTKSFNRGECSGGGGSGGGGSAPLKIQAYFN linkerETADLPCQFANSQNLSLSELVVFWQDQENLVLNEVYLGK (underlined) ERFDSVDSKYMGRTSFDSDSWTLRLHNLQIKDKGRYQCI and CD86 IHHKKPTGMINIHQMNSELSVLA mutant 1040LIGHT CHAIN PREFERABLY ASSEMBLES WITH AHEAVY CHAIN COMPRISING THE 1620 VH  SEQUENCETHUS, COMPLETE MOLECULE MAY BE  DESIGNATED 1620/1767 205 1769 = 1627 ntGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGCGCA Light chain TCTGTAGGAGACCGCGTCACCATCACTTGCCGGGCAAGT VL, with CAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAA constantCCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCC kappaAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGT sequence, GGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTG linkerCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGGGT (underlined) ACTGGTTACGGTCCGCTCACTTTTGGCCAGGGGACCAAG and CD86 CTGGAGATCAAACGTgagtcgtacgctagcaagcttgat mutant 1040atcgaattctaaactctgagggggtcggatgacgtggccattctttgcctaaagcattgagtttactgcaaggtcagaaaagcatgcaaagccctcagaatggctgcaaagagctccaacaaaacaatttagaactttattaaggaatagggggaagctaggaagaaactcaaaacatcaagattttaaatacgcttcttggtctccttgctataattatctgggataagcatgctgttttctgtctgtccctaacatgccctgtgattatccgcaaacaacacacccaagggcagaactttgttacttaaacaccatcctgtttgcttctttcctcagGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTAGCGGAGGAGGAGGAAGCGGAGGAGGAGGAAGCGCCCCCCTCAAAATCCAAGCGTACTTCAACGAAACTGCAGACTTACCGTGTCAGTTTGCCAATTCGCAGAATCTGAGCCTGAGCGAACTGGTGGTTTTCTGGCAGGATCAGGAGAACCTGGTTCTGAACGAAGTCTATCTGGGCAAAGAGCGGTTCGACAGCGTGGACAGCAAGTATATGGGCCGCACCAGCTTTGATAGCGACAGCTGGACCCTGCGTCTGCACAATCTGCAAATCAAAGATAAGGGTAGGTACCAGTGCATTATCCACCATAAGAAGCCGACGGGTATGATTAATATTCACCAAATGAACTCCGAGTTGT CTGTCCTGGCG 206 1769 = 1627 aaDIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQK Light chain PGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSL VL, with QPEDFATYYCQQGTGYGPLTFGQGTKLEIKRTVAAPSVF constantIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQ kappaSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYAC sequence, EVTHQGLSSPVTKSFNRGECSGGGGSGGGGSAPLKIQAY linkerFNETADLPCQFANSQNLSLSELVVFWQDQENLVLNEVYL (underlined) GKERFDSVDSKYMGRTSFDSDSWTLRLHNLQIKDKGRYQ and CD86 CIIHHKKPTGMINIHQMNSELSVLA mutant 1040LIGHT CHAIN PREFERABLY ASSEMBLES WITH AHEAVY CHAIN COMPRISING THE 1626 VH  SEQUENCETHUS, COMPLETE MOLECULE MAY BE  DESIGNATED 1626/1769

TABLE I(1) Exemplary CD137 CDR sequences Antibody CDRH1 CDRH2 CDRH31204/1205 GFTFSSYY IGSYYGYT ARAYYDYNYYYAYFDY (SEQ ID NO: 207)(SEQ ID NO: 212) (SEQ ID NO: 217) 1214/1215 GFTFSSYA IGSGGGYT ARVGHPFDY(SEQ ID NO: 208) (SEQ ID NO: 213) (SEQ ID NO: 218) 1618/1619 GFTFSYGSISSGSGST ARSSYYGSYYSIDY (SEQ ID NO: 209) (SEQ ID NO: 214)(SEQ ID NO: 219) 1620/1621 GFTFSGYY ISSSGSYT ARSVGPYFDY (SEQ ID NO: 210)(SEQ ID NO: 215) (SEQ ID NO: 220) 1626/1627 GFTFGGYS IGGYYYST ARSYYGSIDY(SEQ ID NO: 211) (SEQ ID NO: 216) (SEQ ID NO: 221)

TABLE I(2) Exemplary CD137 CDR sequences Antibody CDRL1 CDRL2 CDRL31204/1205 QSISSY AAS QQSVPHYPFT (SEQ ID NO: 80) (SEQ ID NO: 81)(SEQ ID NO: 222) 1214/1215 QSISSY AAS QQDAYPHT (SEQ ID NO: 80)(SEQ ID NO: 81) (SEQ ID NO: 223) 1618/1619 QSISSY AAS QQYYDNLPT(SEQ ID NO: 80) (SEQ ID NO: 81) (SEQ ID NO: 224) 1620/1621 QSISSY AASQQGVGPYT (SEQ ID NO: 80) (SEQ ID NO: 81) (SEQ ID NO: 225) 1626/1627QSISSY AAS QQGTGYGPLT (SEQ ID NO: 80) (SEQ ID NO: 81) (SEQ ID NO: 226)

Other Sequences

(human CTLA-4) SEQ ID NO: 1MHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIAKEKKPSYNRGLCENAPNRARM (human CD28) SEQ ID NO: 2MLRLLLALNLFPSIQVTGNKILVKQSPMLVAYDNAVNLSCKYSYNLFSREFRASLHKGLDSAVEVCVVYGNYSQQLQVYSKTGFNCDGKLGNESVTFYLQNLYVNQTDIYFCKIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPG PTRKHYQPYAPPRDFAAYRSSEQ ID NO: 3 APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRI HQMNSELSVLASEQ ID NO: 4 MDPQCTMGLSNILFVMAFLLSGAAPLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIP SEQ ID NO: 5APLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVASKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRI HQMNSELSVLA(human CD86) SEQ ID NO: 44MDPQCTMGLSNILFVMAFLLSGAAPLKIQAYFNETADLPCQFANSQNQSLSELVVFWQDQENLVLNEVYLGKEKFDSVHSKYMGRTSFDSDSWTLRLHNLQIKDKGLYQCIIHHKKPTGMIRIHQMNSELSVLANFSQPEIVPISNITENVYINLTCSSIHGYPEPKKMSVLLRTKNSTIEYDGIMQKSQDNVTELYDVSISLSVSFPDVTSNMTIFCILETDKTRLLSSPFSIELEDPQPPPDHIPWITAVLPTVIICVMVFCLILWKWKKKKRPRNSYKCGTNTMEREESEQTKKREKIHIPERSDEAQRVFKSSKTSSCDKSDTCF (murine CTLA-4) SEQ ID NO: 45MACLGLRRYKAQLQLPSRTWPFVALLTLLFIPVFSEAIQVTQPSVVLASSHGVASFPCEYSPSHNTDEVRVTVLRQTNDQMTEVCATTFTEKNTVGFLDYPFCSGTFNESRVNLTIQGLRAVDTGLYLCKVELMYPPPYFVGMGNGTQIYVIDPEPCPDSDFLLWILVAVSLGLFFYSFLVSAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN (murine CD28) SEQ ID NO: 46MTLRLLFLALNFFSVQVTENKILVKQSPLLVVDSNEVSLSCRYSYNLLAKEFRASLYKGVNSDVEVCVGNGNFTYQPQFRSNAEFNCDGDFDNETVTFRLWNLHVNHTDIYFCKIEFMYPPPYLDNERSNGTIIHIKEKHLCHTQSSPKLFWALVVVAGVLFCYGLLVTVALCVIWTNSRRNRLLQVTTMNMTPRRPGLT RKPYQPYAPARDFAAYRP(human OX40) SEQ ID NO: 51MCVGARRLGRGPCAALLLLGLGLSTVTGLHCVGDTYPSNDRCCHECRPGNGMVSRCSRSQNTVCRPCGPGFYNDVVSSKPCKPCTMCNLRSGSERKQLCTATQDTVCRCRAGTQPLDSYKPGVDCAPCPPGHFSPGDNQACKPWTNCTLAGKHTLQPASNSSDAICEDRDPPATQPQETQGPPARPITVQPTEAWPRTSQGPSTRPVEVPGGRAVAAILGLGLVLGLLGPLAILLALYLLRRDQRLPPDAHKPPGGGSFRTPIQEEQADAHSTLAKI SEQ ID NO: 140gcttccacca agggcccatc cgtcttcccc ctggcgccctgctccaggag cacctccgag agcacagccg ccctgggctgcctggtcaag gactacttcc ccgaaccggt gacggtgtcgtggaactcag gcgccctgac cagcggcgtg cacaccttcccggctgtcct acagtcctca ggactctact ccctcagcagcgtggtgacc gtgccctcca gcagcttggg cacgaagacctacacctgca acgtagatca caagcccagc aacaccaaggtggacaagag agttgagtcc aaatatggtc ccccatgcccaccttgccca gcacctgagt tcctgggggg accatcagtcttcctgttcc ccccaaaacc caaggacact ctcatgatctcccggacccc tgaggtcacg tgcgtggtgg tggacgtgagccaggaagac cccgaggtcc agttcaactg gtacgtggatggcgtggagg tgcataatgc caagacaaag ccgcgggaggagcagttcaa cagcacgtac cgtgtggtca gcgtcctcaccgtcctgcac caggactggc tgaacggcaa ggagtacaagtgcaaggtct ccaacaaagg cctcccgtcc tccatcgagaaaaccatctc caaagccaaa gggcagcccc gagagccacaggtgtacacc ctgcccccat cccaggagga gatgaccaagaaccaggtca gcctgacctg cctggtcaaa ggcttctaccccagcgacat cgccgtggag tgggagagca atgggcagccggagaacaac tacaagacca cgcctcccgt gctggactccgacggctcct tcttcctcta cagcaggcta accgtggacaagagcaggtg gcaggagggg aatgtcttct catgctccgtgatgcatgag gctctgcaca accgctacac acagaagagc ctctccctgt ctctgggtaa aSEQ ID NO: 141 agctttctgg ggcaggccgg gcctgacttt ggctgggggcagggaggggg ctaaggtgac gcaggtggcg ccagccaggtgcacacccaa tgcccatgag cccagacact ggaccctgcatggaccatcg cggatagaca agaaccgagg ggcctctgcgccctgggccc agctctgtcc cacaccgcgg tcacatggcaccacctctct tgcagcttcc accaagggcc catccgtcttccccctggcg ccctgctcca ggagcacctc cgagagcacagccgccctgg gctgcctggt caaggactac ttccccgaaccggtgacggt gtcgtggaac tcaggcgccc tgaccagcggcgtgcacacc ttcccggctg tcctacagtc ctcaggactctactccctca gcagcgtggt gaccgtgccc tccagcagcttgggcacgaa gacctacacc tgcaacgtag atcacaagcccagcaacacc aaggtggaca agagagttgg tgagaggccagcacagggag ggagggtgtc tgctggaagc caggctcagccctcctgcct ggacgcaccc cggctgtgca gccccagcccagggcagcaa ggcatgcccc atctgtctcc tcacccggaggcctctgacc accccactca tgctcaggga gagggtcttctggatttttc caccaggctc ccggcaccac aggctggatgcccctacccc aggccctgcg catacagggc aggtgctgcgctcagacctg ccaagagcca tatccgggag gaccctgcccctgacctaag cccaccccaa aggccaaact ctccactccctcagctcaga caccttctct cctcccagat ctgagtaactcccaatcttc tctctgcaga gtccaaatat ggtcccccatgcccaccttg cccaggtaag ccaacccagg cctcgccctccagctcaagg cgggacaggt gccctagagt agcctgcatccagggacagg ccccagccgg gtgctgacgc atccacctccatctcttcct cagcacctga gttcctgggg ggaccatcagtcttcctgtt ccccccaaaa cccaaggaca ctctcatgatctcccggacc cctgaggtca cgtgcgtggt ggtggacgtgagccaggaag accccgaggt ccagttcaac tggtacgtggatggcgtgga ggtgcataat gccaagacaa agccgcgggaggagcagttc aacagcacgt accgtgtggt cagcgtcctcaccgtcctgc accaggactg gctgaacggc aaggagtacaagtgcaaggt ctccaacaaa ggcctcccgt cctccatcgagaaaaccatc tccaaagcca aaggtgggac ccacggggtgcgagggccac acggacagag gccagctcgg cccaccctctgccctgggag tgaccgctgt gccaacctct gtccctacagggcagccccg agagccacag gtgtacaccc tgcccccatcccaggaggag atgaccaaga accaggtcag cctgacctgcctggtcaaag gcttctaccc cagcgacatc gccgtggagtgggagagcaa tgggcagccg gagaacaact acaagaccacgcctcccgtg ctggactccg acggctcctt cttcctctacagcaggctaa ccgtggacaa gagcaggtgg caggaggggaatgtcttctc atgctccgtg atgcatgagg ctctgcacaaccgctacaca cagaagagcc tctccctgtc tctgggtaaatgagtgccag ggccggcaag cccccgctcc ccgggctctcggggtcgcgc gaggatgctt ggcacgtacc ccgtctacatacttcccagg cacccagcat ggaaataaag cacccaccactgccctgggc ccctgtgaga ctgtgatggt tctttccacgggtcaggccg agtctgaggc ctgagtgaca tgagggaggcagagcgggtc ccactgtccc cacactgg SEQ ID NO: 142gcttccacca agggcccatc cgtcttcccc ctggcgccctgctccaggag cacctccgag agcacagccg ccctgggctgcctggtcaag gactacttcc ccgaaccggt gacggtgtcgtggaactcag gcgccctgac cagcggcgtg cacaccttcccggctgtcct acagtcctca ggactctact ccctcagcagcgtggtgacc gtgccctcca gcagcttggg cacgaagacctacacctgca acgtagatca caagcccagc aacaccaaggtggacaagag agttgagtcc aaatatggtc ccccatgcccatcatgccca gcacctgagt tcctgggggg accatcagtcttcctgttcc ccccaaaacc caaggacact ctcatgatctcccggacccc tgaggtcacg tgcgtggtgg tggacgtgagccaggaagac cccgaggtcc agttcaactg gtacgtggatggcgtggagg tgcataatgc caagacaaag ccgcgggaggagcagttcaa cagcacgtac cgtgtggtca gcgtcctcaccgtcctgcac caggactggc tgaacggcaa ggagtacaagtgcaaggtct ccaacaaagg cctcccgtcc tccatcgagaaaaccatctc caaagccaaa gggcagcccc gagagccacaggtgtacacc ctgcccccat cccaggagga gatgaccaagaaccaggtca gcctgacctg cctggtcaaa ggcttctaccccagcgacat cgccgtggag tgggagagca atgggcagccggagaacaac tacaagacca cgcctcccgt gctggactccgacggctcct tcttcctcta cagcaggcta accgtggacaagagcaggtg gcaggagggg aatgtcttct catgctccgtgatgcatgag gctctgcaca accactacac acagaagagc ctctccctgt ctctgggtaa aSEQ ID NO: 143 agctttctgg ggcaggccgg gcctgacttt ggctgggggcagggaggggg ctaaggtgac gcaggtggcg ccagccaggtgcacacccaa tgcccatgag cccagacact ggaccctgcatggaccatcg cggatagaca agaaccgagg ggcctctgcgccctgggccc agctctgtcc cacaccgcgg tcacatggcaccacctctct tgcagcttcc accaagggcc catccgtcttccccctggcg ccctgctcca ggagcacctc cgagagcacagccgccctgg gctgcctggt caaggactac ttccccgaaccggtgacggt gtcgtggaac tcaggcgccc tgaccagcggcgtgcacacc ttcccggctg tcctacagtc ctcaggactctactccctca gcagcgtggt gaccgtgccc tccagcagcttgggcacgaa gacctacacc tgcaacgtag atcacaagcccagcaacacc aaggtggaca agagagttgg tgagaggccagcacagggag ggagggtgtc tgctggaagc caggctcagccctcctgcct ggacgcaccc cggctgtgca gccccagcccagggcagcaa ggcatgcccc atctgtctcc tcacccggaggcctctgacc accccactca tgctcaggga gagggtcttctggatttttc caccaggctc ccggcaccac aggctggatgcccctacccc aggccctgcg catacagggc aggtgctgcgctcagacctg ccaagagcca tatccgggag gaccctgcccctgacctaag cccaccccaa aggccaaact ctccactccctcagctcaga caccttctct cctcccagat ctgagtaactcccaatcttc tctctgcaga gtccaaatat ggtcccccatgcccatcatg cccaggtaag ccaacccagg cctcgccctccagctcaagg cgggacaggt gccctagagt agcctgcatccagggacagg ccccagccgg gtgctgacgc atccacctccatctcttcct cagcacctga gttcctgggg ggaccatcagtcttcctgtt ccccccaaaa cccaaggaca ctctcatgatctcccggacc cctgaggtca cgtgcgtggt ggtggacgtgagccaggaag accccgaggt ccagttcaac tggtacgtggatggcgtgga ggtgcataat gccaagacaa agccgcgggaggagcagttc aacagcacgt accgtgtggt cagcgtcctcaccgtcctgc accaggactg gctgaacggc aaggagtacaagtgcaaggt ctccaacaaa ggcctcccgt cctccatcgagaaaaccatc tccaaagcca aaggtgggac ccacggggtgcgagggccac acggacagag gccagctcgg cccaccctctgccctgggag tgaccgctgt gccaacctct gtccctacagggcagccccg agagccacag gtgtacaccc tgcccccatcccaggaggag atgaccaaga accaggtcag cctgacctgcctggtcaaag gcttctaccc cagcgacatc gccgtggagtgggagagcaa tgggcagccg gagaacaact acaagaccacgcctcccgtg ctggactccg acggctcctt cttcctctacagcaggctaa ccgtggacaa gagcaggtgg caggaggggaatgtcttctc atgctccgtg atgcatgagg ctctgcacaaccactacaca cagaagagcc tctccctgtc tctgggtaaatgagtgccag ggccggcaag cccccgctcc ccgggctctcggggtcgcgc gaggatgctt ggcacgtacc ccgtctacatacttcccagg cacccagcat ggaaataaag cacccaccactgccctgggc ccctgtgaga ctgtgatggt tctttccacgggtcaggccg agtctgaggc ctgagtgaca tgagggaggcagagcgggtc ccactgtccc cacactgg SEQ ID NO: 145gcctccacca agggcccatc ggtcttcccc ctggcaccctcctccaagag cacctctggg ggcacagcgg ccctgggctgcctggtcaag gactacttcc ccgaaccggt gacggtgtcgtggaactcag gcgccctgac cagcggcgtg cacaccttcccggctgtcct acagtcctca ggactctact ccctcagcagcgtggtgacc gtgccctcca gcagcttggg cacccagacctacatctgca acgtgaatca caagcccagc aacaccaaggtggacaagaa agttgagccc aaatcttgtg acaaaactcacacatgccca ccgtgcccag cacctgaact cctggggggaccgtcagtct tcctcttccc cccaaaaccc aaggacaccctcatgatctc ccggacccct gaggtcacat gcgtggtggtggacgtgagc cacgaagacc ctgaggtcaa gttcaactggtacgtggacg gcgtggaggt gcataatgcc aagacaaagccgcgggagga gcagtacaac agcacgtacc gtgtggtcagcgtcctcacc gtcctgcacc aggactggct gaatggcaaggagtacaagt gcaaggtctc caacaaagcc ctcccagcccccatcgagaa aaccatctcc aaagccaaag ggcagccccgagaaccacag gtgtacaccc tgcccccatc ccgggatgagctgaccaaga accaggtcag cctgacctgc ctggtcaaaggcttctatcc cagcgacatc gccgtggagt gggagagcaatgggcagccg gagaacaact acaagaccac gcctcccgtgctggactccg acggctcctt cttcctctac agcaagctcaccgtggacaa gagcaggtgg cagcagggga acgtcttctcatgctccgtg atgcatgagg ctctgcacaa ccactacacgcagaagagcc tctccctgtc tccgggtaaa SEQ ID NO: 146gcctccacca agggcccatc ggtcttcccc ctggcaccctcctccaagag cacctctggg ggcacagcgg ccctgggctgcctggtcaag gactacttcc ccgaaccggt gacggtgtcgtggaactcag gcgccctgac cagcggcgtg cacaccttcccggctgtcct acagtcctca ggactctact ccctcagcagcgtggtgacc gtgccctcca gcagcttggg cacccagacctacatctgca acgtgaatca caagcccagc aacaccaaggtggacaagaa agttggtgag aggccagcac agggagggagggtgtctgct ggaagccagg ctcagcgctc ctgcctggacgcatcccggc tatgcagccc cagtccaggg cagcaaggcaggccccgtct gcctcttcac ccggaggcct ctgcccgccccactcatgct cagggagagg gtcttctggc tttttccccaggctctgggc aggcacaggc taggtgcccc taacccaggccctgcacaca aaggggcagg tgctgggctc agacctgccaagagccatat ccgggaggac cctgcccctg acctaagcccaccccaaagg ccaaactctc cactccctca gctcggacaccttctctcct cccagattcc agtaactccc aatcttctctctgcagagcc caaatcttgt gacaaaactc acacatgcccaccgtgccca ggtaagccag cccaggcctc gccctccagctcaaggcggg acaggtgccc tagagtagcc tgcatccagggacaggcccc agccgggtgc tgacacgtcc acctccatctcttcctcagc acctgaactc ctggggggac cgtcagtcttcctcttcccc ccaaaaccca aggacaccct catgatctcccggacccctg aggtcacatg cgtggtggtg gacgtgagccacgaagaccc tgaggtcaag ttcaactggt acgtggacggcgtggaggtg cataatgcca agacaaagcc gcgggaggagcagtacaaca gcacgtaccg tgtggtcagc gtcctcaccgtcctgcacca ggactggctg aatggcaagg agtacaagtgcaaggtctcc aacaaagccc tcccagcccc catcgagaaaaccatctcca aagccaaagg tgggacccgt ggggtgcgagggccacatgg acagaggccg gctcggccca ccctctgccctgagagtgac cgctgtacca acctctgtcc ctacagggcagccccgagaa ccacaggtgt acaccctgcc cccatcccgggatgagctga ccaagaacca ggtcagcctg acctgcctggtcaaaggctt ctatcccagc gacatcgccg tggagtgggagagcaatggg cagccggaga acaactacaa gaccacgcctcccgtgctgg actccgacgg ctccttcttc ctctacagcaagctcaccgt ggacaagagc aggtggcagc aggggaacgtcttctcatgc tccgtgatgc atgaggctct gcacaaccactacacgcaga agagcctctc cctgtctccg ggtaaa SEQ ID NO: 147cgaactgtgg ctgcaccatc tgtcttcatc ttcccgccatctgatgagca gttgaaatct ggaactgcct ctgttgtgtgcctgctgaat aacttctatc ccagagaggc caaagtacagtggaaggtgg ataacgccct ccaatcgggt aactcccaggagagtgtcac agagcaggac agcaaggaca gcacctacagcctcagcagc accctgacgc tgagcaaagc agactacgagaaacacaaag tctacgcctg cgaagtcacc catcagggcctgagctcgcc cgtcacaaag agcttcaaca ggggagagtg t(Human CD137, amino acid sequence: >gi|571321|gb|AAA53133.1| 4-1BB [Homo sapiens])SEQ ID NO: 148 MGNSCYNIVATLLLVLNFERTRSLQDPCSNCPAGTFCDNNRNQICSPCPPNSFSSAGGQRTCDICRQCKGVFRTRKECSSTSNAECDCTPGFHCLGAGCSMCEQDCKQGQELTKKGCKDCCFGTFNDQKRGICRPWTNCSLDGKSVLVNGTKERDVVCGPSPADLSPGASSVTPPAPAREPGHSPQIISFFLALTSTALLFLLFFLTLRFSVVKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEE GGCEL SEQ ID NO: 175gcttccacca agggcccatc cgtcttcccc ctggcgccctgctccaggag cacctccgag agcacagccg ccctgggctgcctggtcaag gactacttcc ccgaaccggt gacggtgtcgtggaactcag gcgccctgac cagcggcgtg cacaccttcccggctgtcct acagtcctca ggactctact ccctcagcagcgtggtgacc gtgccctcca gcagcttggg cacgaagacctacacctgca acgtagatca caagcccagc aacaccaaggtggacaagag agttgagtcc aaatatggtc ccccatgcccaccttgccca gcacctgagt tcctgggggg accatcagtcttcctgttcc ccccaaaacc caaggacact ctcatgatctcccggacccc tgaggtcacg tgcgtggtgg tggacgtgagccaggaagac cccgaggtcc agttcaactg gtacgtggatggcgtggagg tgcataatgc caagacaaag ccgcgggaggagcagttcaa cagcacgtac cgtgtggtca gcgtcctcaccgtcctgcac caggactggc tgaacggcaa ggagtacaagtgcaaggtct ccaacaaagg cctcccgtcc tccatcgagaaaaccatctc caaagccaaa gggcagcccc gagagccacaggtgtacacc ctgcccccat cccaggagga gatgaccaagaaccaggtca gcctgacctg cctggtcaaa ggcttctaccccagcgacat cgccgtggag tgggagagca atgggcagccggagaacaac tacaagacca cgcctcccgt gctggactccgacggctcct tcttcctcta cagcaggcta accgtggacaagagcaggtg gcaggagggg aatgtcttct catgctccgtgatgcatgag gctctgcaca accactacac acagaagagc ctctccctgt ctctgggtaa aSEQ ID NO: 176 agctttctgg ggcaggccgg gcctgacttt ggctgggggcagggaggggg ctaaggtgac gcaggtggcg ccagccaggtgcacacccaa tgcccatgag cccagacact ggaccctgcatggaccatcg cggatagaca agaaccgagg ggcctctgcgccctgggccc agctctgtcc cacaccgcgg tcacatggcaccacctctct tgcagcttcc accaagggcc catccgtcttccccctggcg ccctgctcca ggagcacctc cgagagcacagccgccctgg gctgcctggt caaggactac ttccccgaaccggtgacggt gtcgtggaac tcaggcgccc tgaccagcggcgtgcacacc ttcccggctg tcctacagtc ctcaggactctactccctca gcagcgtggt gaccgtgccc tccagcagcttgggcacgaa gacctacacc tgcaacgtag atcacaagcccagcaacacc aaggtggaca agagagttgg tgagaggccagcacagggag ggagggtgtc tgctggaagc caggctcagccctcctgcct ggacgcaccc cggctgtgca gccccagcccagggcagcaa ggcatgcccc atctgtctcc tcacccggaggcctctgacc accccactca tgctcaggga gagggtcttctggatttttc caccaggctc ccggcaccac aggctggatgcccctacccc aggccctgcg catacagggc aggtgctgcgctcagacctg ccaagagcca tatccgggag gaccctgcccctgacctaag cccaccccaa aggccaaact ctccactccctcagctcaga caccttctct cctcccagat ctgagtaactcccaatcttc tctctgcaga gtccaaatat ggtcccccatgcccaccttg cccaggtaag ccaacccagg cctcgccctccagctcaagg cgggacaggt gccctagagt agcctgcatccagggacagg ccccagccgg gtgctgacgc atccacctccatctcttcct cagcacctga gttcctgggg ggaccatcagtcttcctgtt ccccccaaaa cccaaggaca ctctcatgatctcccggacc cctgaggtca cgtgcgtggt ggtggacgtgagccaggaag accccgaggt ccagttcaac tggtacgtggatggcgtgga ggtgcataat gccaagacaa agccgcgggaggagcagttc aacagcacgt accgtgtggt cagcgtcctcaccgtcctgc accaggactg gctgaacggc aaggagtacaagtgcaaggt ctccaacaaa ggcctcccgt cctccatcgagaaaaccatc tccaaagcca aaggtgggac ccacggggtgcgagggccac acggacagag gccagctcgg cccaccctctgccctgggag tgaccgctgt gccaacctct gtccctacagggcagccccg agagccacag gtgtacaccc tgcccccatcccaggaggag atgaccaaga accaggtcag cctgacctgcctggtcaaag gcttctaccc cagcgacatc gccgtggagtgggagagcaa tgggcagccg gagaacaact acaagaccacgcctcccgtg ctggactccg acggctcctt cttcctctacagcaggctaa ccgtggacaa gagcaggtgg caggaggggaatgtcttctc atgctccgtg atgcatgagg ctctgcacaaccactacaca cagaagagcc tctccctgtc tctgggtaaatgagtgccag ggccggcaag cccccgctcc ccgggctctcggggtcgcgc gaggatgctt ggcacgtacc ccgtctacatacttcccagg cacccagcat ggaaataaag cacccaccactgccctgggc ccctgtgaga ctgtgatggt tctttccacgggtcaggccg agtctgaggc ctgagtgaca tgagggaggcagagcgggtc ccactgtccc cacactgg

EXAMPLES

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting. The contents of allfigures and all references, patents and published patent applicationscited throughout this application are expressly incorporated herein byreference.

Example 1—CTLA-4 Binding Domains

CTLA-4 binding domain polypeptides were selected and expressed asdescribed in WO 2014/207063 (see Examples) and were assayed for bindingto CTLA-4 by at least one of ELISA and surface plasmon resonance asdescribed below.

Binding ELISA

96-well flat bottom high binding plates (Greiner, #655074) were coatedwith either CTLA4-Fc (Fitzgerald, #30R-CD152) or CD28-Fc (R&D systems,342-CD) by incubating overnight at 4° C. The plates were washed (Washbuffer: PBS+0.05% Tween 20 (PBST) Medicago, #09-9410-100) and thenblocked in PBST+3% BSA (Merck, #1.12018.0100). The plates were washedagain and sample or controls (serially diluted 1/5 from 200-0,001 μg/ml)were added to the wells. The samples were incubated for 1 h at roomtemperature and then washed. Detection antibody, goat-anti-human IgGFcγ-HRP (Jackson, #109-035-098) was added and the plates weresubsequently developed using SuperSignal Pico Chemiluminescent substrate(Thermo Scientific, #37069) and detected with an Envision reader (PerkinElmer). EC50 values were calculated for both CTLA4 and CD28. The bindingratio (EC50 binding ratio=[EC50 for CD28]÷[EC50 for CTLA-4]) wascalculated for each polypeptide and is shown in Table 1.1.

Surface Plasmon Resonance

Either CTLA4-Fc (Fitzgerald, #30R-CD152) or CD28-Fc (R&D Systems,342-CD) was immobilized to the Biacore™ sensor chip, CMS, usingconventional amine coupling. The CD86 mutant molecules and controls(serially diluted 1/2 100-1.5 nM) were analyzed for binding in HBS-P(GE, BR-1003-68) at a flow rate of 30 μl/ml. The association wasfollowed for 3 minutes and the dissociation for 10 minutes. Regenerationwas performed twice using 5 mM NaOH for 30 seconds. The kineticparameters and the affinity constants were calculated usingBIAevaluation 4.1 software (Table 1.3).

Inhibition ELISA

96-well flat bottom plate high binding plates (Greiner, #655074) werecoated with wildtype CD86-Fc (R&D Systems, #7625-B2) by incubatingovernight at 4° C. The plates were washed (Wash buffer: PBS+0.05% Tween20 (PBST) Medicago, #09-9410-100) and then blocked in PBST+3% BSA(Merck, #1.12018.0100). The sample (CD86 mutant or wild type protein;serially diluted 1/4 from 30000 to 0.3 ng/ml) was incubated withbiotinylated-CTLA4 (Fitzgerald, #30R-CD152) in room temperature 1 h, themixture was then added to the blocked wells in the ELISA plate.Detection was performed with Streptavidin-HRP (Pierce, #21126) and theplates were subsequently developed using SuperSignal PicoChemiluminescent substrate (Thermo Scientific, #37069) and detected withEnvision reader (Perkin Elmer). The results are shown in FIG. 2. IC50values were calculated and are shown in the tables below. All moleculestested showed better IC50 value than both wild type and H79A, the IC ofthe best mutant CD86 molecule was improved over 100-fold compared towild type. Results for exemplary molecules 900, 901, 904, 906, 907, 908,910, 915 and 938 are shown in Table 1.1. Kd binding ratio=[Kd forCD28]÷[Kd for CTLA-4]. The full amino acid sequences for exemplarymolecules 900, 901, 904, 906, 907, 908, 910, 915 and 938 are provided asSEQ ID NOs: 6 to 14, respectively.

TABLE 1.1 Mutated positions and amino acid change EC50 Kd Sam- relativeto wild-type binding binding ple (positions numbered as in FIG. 4) ratioratio 900 H79D, L107I, I111V, T118S, M120L, 3.5 ND* I121V, R122K, Q125E901 Q48L, S49T, L107I, I111V, R122K, 17.2 2.7 Q125E, A134T 904 V54I,K74R, S77A, H79D, L107I, 12.2 6.8 T118S, I121V, R122K, N127D 906 F32I,Q48L, K74R, H79D, L107F, 16.2 0.8 M120L, I121V, R122K, Q125E 907 R122K,Q125E 30.5 5.6 908 L107I, I121V, R122K, Q125E 6.2 4.7 910 H79D, L107I,I121V, R122K, Q125E 7.7 5.1 915 V64I, H79D, L107F, T118S, M120L, 9.9 1.9R122K, N127D 938 V64I, L107I, I121V, R122K 2.0 5.5 Wild type 3.4 1.6 *Nodetectable binding was seen in the BIAcore ™ analysis nor binding ELISA

Results for exemplary molecules 1038, 1039, 1040, 1041,1042, 1043, 1044,1045, 1046 and 1047 are shown in Tables 1.2 and 1.3, and in FIGS. 2 and3. The full amino acid sequences for exemplary molecules 1038, 1039,1040, 1041, 1042, 1043, 1044, 1045, 1046 and 1047 are provided as SEQ IDNOs: 15 to 24, respectively.

TABLE 1.2 Sample EC50 Sample IC50 1038 0.14 — — 1039 0.039 — — 10400.0076 1040 0.049 1041 0.087 1041 3.1 1042 0.29 1042 4.3 1043 0.035 10434.0 1044 0.029 1044 1.4 1045 0.047 1045 2.6 1046 0.019 1046 1.1 10470.037 1047 0.98 Wild type 0.51 Wild type 15 Prior Art 0.81 H79A 25Negative No Negative No control activity control activity

TABLE 1.3 Mutated positions and Sam- amino acid change ka kd K_(D) ple(positions numbered as in FIG. 4) (1/Ms) (1/s) (nM) 1038 Q48L, H79D,L107I, I121V, 1.0e6 0.012 12 R122K, Q125E 1039 Q48L, H79S, L107I, T118S,1.0e6 8.5e−3 8 I121V, R122K, N127D 1040 Q48L, K74R, H79D, L107R, R122N1.0e6 3.2e−3 3 1041 F32L, Q48L, H79D, L107I, 7.0e5 8.4e−3 12 M120L,I121V, R122K, 125E 1042 Q48L, K74I, H79S, L107I, 4.4e5 0.011 25 T118S,I121V, R122K, N127D 1043 Q48L, H79D, L107I, R122K, Q125E 1.1e6 0.011 101044 Q48L, S49T, H79S, L107I, 1.1e6 9.4e−3 8 R122K, Q125E, N127S 1045Q48L, S49T, H79D, M120L, 9.4e5 8.3e−3 9 I121V, R122K, Q125E 1046 H79D,K103E, L107I, T118S, 1.4e6 8.0e−3 6 I121V, R122K, N127D 1047 Q48L, H79D,L107I, M120L, 8.5e5 8.4e−3 10 I121V, R122K, Q125E Wild 4.6e5 0.023 50type H79A 3.4e5 0.022 63

Example 2—Cross Reactivity to Murine CTLA-4 of Exemplary Polypeptidefrom Clone 1040

The relative affinity for murine and human CTLA-4 of an exemplary mutantCD86 molecule 1040 was investigated using an inhibition ELISA bindingassay. The 1040 molecule used in these experiments was conjugated to ananti-CD40 antibody as part of a bispecific molecule. The CTLA-4 bindingproperties of the CD86 molecule are not affected by this conjugation(data not shown).

In brief, 96-well flat bottom plate high binding plates (Greiner#655074) were coated with human CTLA-4 (Fitzgerald) incubating overnightat 4° C. The plates were washed (Wash buffer: PBS+0.05% Tween 20 (PBST)Medicago #09-9410-100) and then blocked in PBST+3% BSA (Merck,#1.12018.0100).

The sample (exemplary CD86 mutant) was pre-incubated at room temperaturefor 1 hour with soluble biotinylated human CTLA4 (Fitzgerald #30R-CD152)or soluble murine CTLA-4 (R&D systems) at different concentrations(serial dilutions 1/4 from 30000 to 0.3 ng/ml).

The mixture was then added to the blocked wells in the ELISA plate.Detection was performed with Streptavidin-HRP (Pierce, #21126) and theplates were subsequently developed using SuperSignal PicoChemiluminescent substrate (Thermo Scientific, #37069) and detected withEnvision reader (Perkin Elmer). The results are shown in FIG. 5. Theobserved inhibition curves with murine and human CTLA-4 demonstrate thatthe binding affinity of the exemplary CD86 mutant (1040) to the twoforms of CTLA-4 is of a similar magnitude. The other clones tested inExample 1 were also found to bind to murine CTLA-4 (data not shown).

Example 3—Characterisation of OX40 Antibodies

Characteristics of exemplary OX40 antibodies are summarised in Table 3.1below.

TABLE 3.1 Dose huOX40 Dissociation Association Association CDR H3response M. mulatta binding constant rate constant rate constant lengthELISA T-cell OX40 FACS Hydrophathy Isoelectric K_(D) ka kd Antibody(IMGT) (IgG) agonist binding (CHO) index point (M) (1/Ms) (1/s)1166/1167 10 <1 nM Yes Yes Yes −0.392 9.11 3.22E−10 9.01E+04 2.90E−051170/1171 10 <1 nM Yes Yes Yes −0.415 9.11 2.50E−10 1.45E+06 3.63E−041164/1135 11 <1 nM Yes Yes Yes −0.398 9.21 3.08E−10 2.51E+05 7.73E−051168/1135 11 <1 nM Yes Yes Yes −0.404 9.19 3.27E−10 5.18E+05 1.69E−041482/1483 9 <1 nM Yes Yes Yes −0.381 9.19 7.53E−10 7.76E+05 5.84E−041490/1135 11 <1 nM Yes Yes Yes −0.407 9.18 3.07E−10 3.87E+06 1.19E−031514/1515 14 <1 nM Yes Yes Yes −0.399 9.11 6.40E−10 2.57E+05 1.64E−041520/1135 17 <1 nM Yes Yes Yes −0.394 9.18 5.55E−10 6.20E+05 3.44E−041524/1525 10 <1 nM Yes Yes Yes −0.391 9.11 8.11E−10 1.71E+06 1.39E−031526/1527 15 <1 nM Yes Yes Yes −0.388 8.99 4.30E−10 4.35E+05 1.87E−041542/1135 11 <1 nM Yes Yes Yes −0.411 9.2 4.63E−10 2.16E+05 1.00E−04

Two anti-OX40 antibodies were synthesised solely for use as referenceantibodies for the purposes of comparison in these studies. They aredesignated herein as “72” or “72/76”, and “74” or “74/78”, respectively.

Measurement of Kinetic Constants by Surface Plasmon Resonance

Human OX40 (R&D systems, #3358_OX) was immobilized to the Biacore™sensor chip, CMS, using conventional amine coupling. The testedantibodies and controls (serially diluted 1/3 or 1/2 100-2 nM) wereanalyzed for binding in HBS-P (GE, # BR-1003-68) at a flow rate of 30μl/ml. The association was followed for 3 minutes and the dissociationfor 20 minutes. Regeneration was performed twice using 50 mM NaOH for 60seconds. The kinetic parameters and the affinity constants werecalculated using 1:1 Langmuir model with drifting baseline. The testedantibodies were overall in the subnanomolar-nanomolar range with varyingon and off rates (FIG. 6 and Table 3.1). Most of the antibodies hadaffinities <5 nM. The kinetic parameters and the affinity constants werecalculated using BIAevaluation 4.1 software.

Measurement by ELISA of Binding to Human and Murine OX40, and to CD137and CD40 by ELISA

ELISA plates were coated with human OX40 (R&D Systems, 3388-OX), CD40(Ancell), or CD137 (R&D Systems) at 0.1 or 0.5 μg/ml. The ELISA plateswere washed with PBST and then blocked with PBST+2% BSA for 1 h at roomtemperature and then washed again with PBST. The antibodies were addedin dilution series to the ELISA plates for 1 h at room temperature andthen washed with PBST. Binding was detected using goat anti human kappalight chain HRP, incubated for 1 h at room temperature. SuperSignal PicoLuminescent was used as substrate and luminescence was measured usingFluostar Optima.

All the tested OX40 antibodies bound to human OX40 and displayed EC50value below 1 nM. The antibodies did not bind to murine OX40 or to theother TNFR super family members tested (data not shown).

Measurement of Binding to Human OX40 Over-Expressed on CHO Cells

The extracellular part of human OX40 was fused to the transmembrane andintracellular part of hCD40 and cloned into pcDNA3.0. The vector wassubsequently stably transfected into CHO cells. Expression of OX40 wasconfirmed by incubating with commercial OX40 antibody (huOX40, BDBiosciences) for 30 min at 4° C. and then detected with a-huIgG-PE(Jackson Immunoresearch) 30 min 4° C. For the assay, the transfectedcells were incubated with the test antibodies and controls for 30 min at4° C. and then detected with a-huIgG-PE (Jackson Immunoresearch) 30 min4° C. Cells were analyzed by flow cytometry with FACS Verse (BDBiosciences).

All clones bound to hOX40 overexpressed on CHO cells in a dose dependentmanner (FIG. 7).

Example 4—Sequence Analysis of OX40 Antibodies

The CDR sequences of both the heavy and light chain variable regionswere analysed for each antibody. Table 4.1 illustrates the analysis asconducted for the VH CDR3 sequences. Positions in Table 4.1 are definedaccording to IMGT numbering system. The following patterns wereidentified.

The VH regions all comprise:

(a) a heavy chain CDR1 sequence which is 8 amino acids in length andcomprises the consensus sequence: “G, F, T, F, G/Y/S, G/Y/S, Y/S,Y/S/A”;

(b) a heavy chain CDR2 sequence which is 8 amino acids in length andcomprises the consensus sequence: “I, G/Y/S/T, G/S/Y, S/Y, G/S/Y, G/S/Y,G/S/Y, T”; and

(c) a heavy chain CDR3 sequence which is 9 to 17 amino acids in lengthand which comprises the consensus sequence of: “A, R, G/Y/S/H,G/Y/F/V/D, G/Y/P/F, −/H/S, −/N/D/H, −/Y/G, −/Y, −/Y, −/W/A/V, −/A/Y,−/D/A/Y/G/H/N, Y/S/W/A/T, UM/I/F, D, Y”.

The VL regions all comprise:

(a) a light chain CDR1 sequence which consists of the sequence: “Q, S,I, S, S, Y”;

(b) a light chain CDR2 sequence which consists of the sequence: “A, A,S”;

(c) a light chain CDR3 sequence which is 8 to 10 amino acids in lengthand comprises the consensus sequence: “Q,Q, S/Y/G, −/Y/H/G, −/S/Y/G/D,S/Y/G/D, S/Y/G/T, P/L, Y/S/H/L/F, T”.

Within the consensus sequence for the heavy chain CDR3, two sub-familieswere identified. Each antibody in the first sub-family comprises a VHCDR3 sequence of 10 amino acids in length which comprises the consensussequence “A, R, Y/H, D, Y, A/Y/G, S/W/A, M/L, D, Y”. Antibodies in thisfamily are referred to as family Z and are identified as such in Table4.1. Each antibody in the second sub-family comprises a VH CDR3 sequenceof 11 amino acids in length which comprises the consensus sequence “A,R, G/Y, V/F/Y, P, H, G/Y/H, Y, F/I, D, Y”. Antibodies in this family arereferred to as family P and are identified as such in Table 4.1.Antibodies of family Z or P are preferred. Antibodies having a VHsequence in family P typically also include a VL sequence with a CDR3sequence of “Q, Q, S, Y, S, T, P, Y, T”, a CDR1 sequence “Q,S,I,S,S,Y”and a CDR2 sequence of “A,A,S”. Accordingly antibodies with a VL regioncomprising these three CDR sequences are preferred.

TABLE 4.1 CDRH3 FAM- VH 105 106 107 108 109 110 111 111.1 111.2 112.2112.1 112 113 114 115 116 117 LENGTH ILY 1482 A R G Y G Y L D Y 9 1166 AR Y D Y A S M D Y 10 Z 1170 A R Y D Y Y W M D Y 10 Z 1524 A R H D Y G AL D Y 10 Z 1164 A R G V P H G Y F D Y 11 P 1168 A R Y F P H Y Y F D Y 11P 1490 A R Y Y P H H Y I D Y 11 P 1542 A R G Y P H H Y F D Y 11 P 1514 AR S G Y S N W A N S F D Y 14 1526 A R Y Y F H D Y A A Y S L D Y 15 1520A R Y Y Y S H G Y Y V Y G T L D Y 17

Example 5—Domain Mapping of OX40 Antibodies

The extracellular part of OX40 consists of four domains, each of whichcan be subdivided into two modules. Genes of OX40 human/mouse chimeraswere synthesized using standard laboratory techniques. The differentchimeras were designed by exchanging domains or modules of the humanOX40 with corresponding mouse OX40. The chimeras were designed based onevaluation of the human and mouse sequences and 3D investigation ofhuman OX40. The synthesized genes were assigned project specific IDnumbers (see Table 5.1). The constructs were cloned into pcDNA3.1 vector(Invitrogen).

The mouse/human chimeras were transiently transfected into FreeStyle293-F cells (Invitrogen), incubated 48 hours in FreeStyle 293 expressionmedium (Invitrogen) 37 C, 8% CO₂, 135 rpm. The transfected cells wereincubated with human OX40 antibodies, human OX40L (hOX40L, RnD Systems),mouse OX40L (mOX40L, RnD Systems) and controls for 30 min 4° C. and thendetected with a-huIgG-PE (Jackson Immunoresearch) 30 min 4° C. Cellswere analyzed with FACS Verse (BD Biosciences). Binding to the differentchimeric constructs were calculated as relative (mean fluorescenceintensity) MFI compared to the binding of the isotype control. Resultsare shown in Table 5.2.

None of the human OX40 antibodies tested bind to murine OX40.Accordingly, if a given antibody does not bind to a particular chimera,this indicates that the antibody is specific for one of the domainswhich has been replaced with a murine domain in that chimera.

TABLE 5.1 Identity of chimeric constructs ID Description of codingregion construct of the chimeric DNA constructs 1544 Human OX40 withmouse domains 1A, 1B and 2A (aa 30-81) 1545 Human OX40 with mousedomains 1B, 2A and 2B (aa 43-107) 1546 Human OX40 with mouse domains 2A,2B and module 3 (aa 66-126) 1547 Human OX40 with mouse domain 2B, module3 and domain 4A (aa 83-141) 1548 Human OX40 with mouse module 3 anddomains 4A and 4B (aa 108-167) 1549 Human OX40 with mouse domains 1A and4B and region non-annotated extracellular region (aa 30-65 and aa127-214) 84 Construct containing the full length OX40 sequence 57 Emptyvector (negative control)

At least four separate binding patterns were identified.

Pattern A:

Antibodies 1170/1171, 1524/1525, and 1526/1527 display a similar bindingpattern and depend on residues in the same domains for binding. Aminoacid residues critical for binding are likely located in module B indomain 2, and in module A of domain 2. The majority of the antibodieswith CDRH3 family “Z” bind according to pattern A (1166/1167 being theexception), indicating that antibodies with this type of CDRH3 arepredisposed to bind this epitope.

Pattern B:

Antibodies 1168/1135, 1542/1135, 1520/1135, 1490/1135, 1482/1483 and1164/1135 display a similar binding pattern and depend mainly onresidues located in Domain 3 for binding. All antibodies with CDRH3family “P” binds with this pattern, demonstrating that the similarity inCDRH3 sequence reveals a common binding epitope.

Pattern C:

Antibody 1166/1167 has a unique binding pattern and likely depends onresidues located in module A and module B in domain 2 for binding.However, both modules must be exchanged simultaneously to abolishbinding, suggesting a structurally complex epitope.

Pattern D:

Antibody 1514/1515 displays a unique binding profile and likely dependsmostly on amino acids located in module B in domain 2 for binding.

Reference antibody 72 binds according to pattern B. The binding patternof the human OX40 ligand is similar to pattern C.

TABLE 5.2 Results from domain mapping experiment Chimeric Antibody OX401490/ 1170/ 1524/ 1526/ 1482/ 1514/ 1164/ 1168/ 1520/ 1542/ 1166/polyclonal construct 1135 1171 1525 1527 1483 1515 1135 1135 1135 11351167 antibody 1544 4.2 2.0 1.5 1.1 8.4 11.6 14.0 13.2 9.4 7.1 14.2 50.91545 28.9 1.0 0.9 1.3 44.4 1.2 37.3 46.6 32.6 40.2 1.0 58.6 1546 1.0 0.80.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.8 30.0 1547 1.1 2.3 1.0 1.0 1.0 1.11.0 1.0 0.9 1.0 15.2 43.9 1548 1.1 20.3 15.4 12.7 1.1 17.2 1.0 1.0 1.11.2 15.8 31.7 1549 5.9 12.1 11.2 8.5 8.7 10.6 14.1 15.1 8.5 13.0 11.326.3 84 14.2 33.4 31.4 21.4 24.9 25.9 27.5 29.6 25.4 29.4 27.6 53.2 571.0 1.0 1.0 1.0 1.0 1.1 1.0 0.9 1.0 1.1 1.0 1.2

Example 6—Cross Reactivity with Macaca mulatta

The extracellular part of OX40 from Macaca mulatta was fused to thetransmembrane and intracellular part of hCD40 and cloned into pcDNA3.0.The vector was subsequently stably transfected into HEK cells(macOX40-HEK).

Expression of OX40 was confirmed by incubating with commercial OX40antibody (huOX40, BD Biosciences) for 30 min at 4° C. and then detectedwith a-huIgG-PE (Jackson Immunoresearch) 30 min 4° C. For the assay, thetransfected cells were incubated with the test antibodies and controlsfor 30 min at 4° C. and then detected with a-huIgG-PE (JacksonImmunoresearch) 30 min 4° C. Cells were analyzed by flow cytometry withFACS Verse (BD Biosciences).

As shown in Table 6.1 below, tested antibodies bind to Macaca mulattaOX40 with EC50 values comparable to those achieved for human OX40,suggesting that Macaca mulatta will be suitable for use in toxicologystudies.

Macaca mulatta is genetically very similar to Macaca fascicularis(cynomolgus monkey) making it very likely that cynomolgus monkey is alsoa suitable species for toxicology studies.

TABLE 6.1 Binding to human and monkey OX40 (95% confidence intervals)OX40 Binding to M mulatta Binding to human antibody OX40, EC50 (μg/mL)OX40, EC50 (μg/mL) 1166/1167 0.1595 to 0.2425 0.1415 to 0.2834 1168/11350.09054 to 0.1939 0.06360 to 0.1308 1482/1483 0.1565 to 0.3120 0.08196to 0.1822 1520/1135 0.1632 to 0.3587 0.09247 to 0.2749 1526/1527 0.2921to 0.5888 0.1715 to 0.4292 1542/1135 0.7221 to 1.414 0.3223 to 0.5525

Example 7—Agonistic Activity in a Human T Cell Assay

Human T cells were obtained by negative T cell selection kit fromMiltenyi from PBMC from leucocyte filters obtained from the blood bank(Lund University Hospital). The OX40 antibodies were coated to thesurface of a 96 well culture plate (Corning Costar U-shaped plates(#3799) and cultured with a combination of immobilized anti-CD3 antibody(UCHT1), at 3 μg/ml, and soluble anti-CD28 antibody (CD28.2), at 5μg/ml. Anti-CD3 was pre-coated overnight at 4° C. On the following day,after one wash with PBS, the OX40 antibodies were coated 1-2 h at 37° C.After 72 h incubation in a moisture chamber at 37° C., 5% CO₂ the IL-2levels in the supernatant were measured.

The ability of the antibodies to stimulate human T cells to produce IL-2was compared with the reference antibody 74 and the relative activity isdisplayed in FIG. 8. The majority of the antibodies provided T cellactivation levels that were comparable with the reference antibody. Anumber of antibodies provided higher levels of T cell activation.

Bispecific Molecules

In the following Examples, tested bispecific molecules are referred toby number, e.g. 1164/1141. This means that the molecule comprises theamino acid sequences of the respective VH and VL regions shown in TablesB and D. For example, 1164/1141 comprises the heavy chain VH regionsequence 1164 shown in Table B (SEQ ID NO: 99), and the bispecific chainnumber 1141 shown in Table D (SEQ ID NO: 129). The specified VH regionsequence of a given molecule is typically provided linked (as part of asingle contiguous polypeptide chain) to the IgG1 heavy chain constantregion sequence of SEQ ID NO: 135. This sequence is typically present atthe C terminal end of a specified VH region sequence of Table B.

Example 8—Affinity of Exemplary Bispecific Molecules for Binding toSingle Targets

Measurement of Kinetic Constants by Surface Plasmon Resonance

Human OX40 (R&D systems, #3358_OX) was immobilized to the Biacore™sensor chip, CMS, using conventional amine coupling. The testedantibodies and controls (serially diluted 1/3 or 1/2 100-2 nM) wereanalyzed for binding in HBS-P (GE, # BR-1003-68) at a flow rate of 30μl/ml. The association was followed for 3 minutes and the dissociationfor 20 minutes. Regeneration was performed twice using 50 mM NaOH for 30seconds. The kinetic parameters and the affinity constants werecalculated using 1:1 Langmuir model with drifting baseline. The testedmolecules had varying on and off rates with generally lower affinity forOX40 than the corresponding monomeric antibodies, but were still in thenanomolar range (Table 8.1).

TABLE 8.1 BsAb ka (1/Ms) kd (1/s) KD (nM) 1164/1141 8.87E+04 1.72E−041.94 1168/1141 2.84E+05 3.05E−04 1.07 1166/1261 7.04E+04 1.12E−04 1.591170/1263 5.18E+05 6.39E−04 1.23

Measurement by ELISA

ELISA plates were coated with human with CTLA-4 (BMS, Orencia) or humanOX40 (R&D Systems, 3388-OX) at 0.4 or 0.5 μg/ml, respectively. The ELISAplates were washed with PBST and then blocked with PBST+2% BSA for 1 hat room temperature and then washed again with PBST. The bispecificmolecules were added in dilution series to the plates and incubated for1 h at room temperature. The ELISA plates were washed, and binding wasdetected using goat anti human kappa light chain HRP for 1 h at roomtemperature. SuperSignal Pico Luminescent was used as substrate andluminescence was measured using Fluostar Optima.

All the tested bispecific molecules bound to both targets and the EC50values are in the range that would be expected based on their affinityas monospecific antibodies (FIG. 9).

Example 9—Dual Binding to Both Targets of Exemplary Bispecific Molecules

Measurement by Surface Plasmon Resonance

Human OX40 (R&D systems, #3358_OX) was immobilized to the Biacore™sensor chip, CMS, using conventional amine coupling. The testedbispecific molecules (0.5 μM or 0.25 μM) and controls were run over thechip at a flow rate of 30 μl/ml. The association was followed for 3minutes and the dissociation for 3 minutes. CTLA4-Fc (BMS, Orencia) wasthen injected and association followed for 3 minutes and thedissociation for 3 minutes. As a control a blank PBS was injectedinstead of CTLA4.

All the tested bispecific molecules bound to both targetssimultaneously, as is shown in FIG. 10).

Measurement by ELISA

ELISA plates were coated with OX40-Fc (R&D systems, #3358_OX) (0.4μg/ml) over night at 4° C. The ELISA plates were washed with PBST andthen blocked with PBST+2% BSA for 1 h at room temperature and thenwashed again with PBST. Bispecific molecules were added in dilutions tothe plates and incubated for 1 h at room temperature. The ELISA plateswere washed and biotinylated CTLA-4 (1 μg/ml) was added and incubated onthe plates at room temperature. The plates were washed and HRP-labelledstreptavidin was used for detection of binding. SuperSignal PicoLuminescent was used as substrate and luminescence was measured usingFluostar Optima.

Binding to both targets was confirmed for all tested bispecificmolecules. As is shown in FIG. 11, the tested bispecific molecules couldbe detected at a concentration 0.1 nM, which corresponds to 0.015 μg/ml.The relative values in the assay correspond well to the affinitiesmeasured by surface plasmon resonance.

Example 10—Agonistic Activity of Exemplary Bispecific Molecules in aHuman CD4 T Cell Assay

Human CD4 T cells were isolated by negative CD4 T cell selection(Miltenyi, human CD4+ T cell Isolation Kit 130-096-533) of PBMC fromleucocyte filters obtained from the blood bank (Lund UniversityHospital). CTLA-4 (Orencia, 2.5 μg/ml) and anti-CD3 (UCHT-1, 1 ug/ml)was coated to the surface of a 96-well culture plate (non-tissuecultured treated, U-shaped 96-well plates (Nunc, VWR #738-0147) overnight at 4° C. By coating with both CTLA-4 and CD3, the assay providesan experimental model of a tumor microenvironment with over-expressedCTLA-4. CTLA-4 was omitted from some wells as a control.

Bispecific molecules to be tested were added soluble in a serialdilution to the wells and compared at the same molar concentrations withcontrols. Two different controls were used for each bispecific moleculetested. The first control is a bispecific molecule designated 1756/1757(an isotype control antibody fused to the 1040 CTLA4 bindingregion=binds CTLA4 but not OX40). The second control is a mixture of thebispecific 1756/1757 control and the monospecific OX40 antibody, whichcorresponds to the tested bispecific molecule. After 72 h of incubationin a moisture chamber at 37° C., 5% 002, IL-2 levels were measured inthe supernatant.

As shown in FIG. 12, there is a dose-dependent effect of the bispecificmolecules, which induce an increase in human T cell activation (measuredby an increase in IL-2 production) when cultured in plates coated withCTLA-4. The controls do not. FIG. 13 shows the results of the same assaywhen conducted at a fixed concentration for the bispecific antibodiesand controls (1.5 nM) in the presence or absence of CTLA-4. The increasein T cell activation is not seen in the absence of CTLA-4. The foldchange in IL-2 levels induced by each bispecific molecule compared tothe corresponding combination of monospecific molecules is shown inTable 10.1. The mean value of IL-2 produced (pg/ml) for each bispecificmolecule or control is shown in Table 10.2.

Since this assay represents an experimental model of a tumormicroenvironment with over-expressed CTLA-4, the results suggest thatthe tested bispecific molecules can be expected to have a greater effectthan monospecific antibodies in such a microenvironment.

TABLE 10.1 Fold change in IL-2 level induced by bispecific moleculecompared to the corresponding 1164/1141 1166/1261 1168/1141 1170/11411514/1581 1520/1141 7.6 7.8 7.2 4.7 5.5 1.7

combination of the monospecific molecules at 1.5 nM

TABLE 10.2 Mean IL-2 level (pg/ml) induced at 1.5 nM bispecific antibodyor control 1164/1135 + 1166/1167 + 1168/1135 + 1170/1171 + 1164/11411166/1261 1756/1757 1756/1757 1168/1141 1170/1141 1756/1757 1756/17571514/1581 1520/1141 Mean 5024 4292 665 550 2681 2575 371 552 5109 1303SD 1058 1333 273 456 954 992 162 285 2600 812 1514/1515 + 1520/1135 +1526/1527 + 1542/1543 + 1756/1757 1756/1757 1526/1585 1542/11411756/1757 1756/1757 1756/1757 Mean 927 774 3200 2671 746 697 1047 SD 653451 1350 1016 346 418 601

Example 11—Stability of Exemplary Bispecific Molecules

The melting point of the antibodies was analyzed by differentialscanning fluorimetry (DSF). Antibody samples in PBS were mixed withSYPRO Orange which was diluted 1000-fold. Thermal scanning between 25and 95° C. was performed in a real-time PCR machine with measurementsmade each degree. A reference antibody 250/251 was used for comparisonand the difference in melting temperature Tm (ΔT_(m)) relative to thereference was determined. T_(m) differences of more than 1.1° C.compared to the reference are considered statistically significant. Asshown in Table 11.1, all tested bispecific molecules displayed goodthermostability with values of 65° C. or above.

TABLE 11.1 Melting temperature Antibody (° C.) 1168/1141 65.6 1164/114165.5 1160/1259 68.5 1166/1261 67.8 1170/1263 66.4 1514/1581 65.31520/1141 65.0 1526/1585 67.6 1542/1141 66.3

Example 12—Characterisation of CD137 Antibodies

General chemical properties of an exemplary CD137 antibody (referred toas 1204/1205) were determined by routine analysis and are shown in Table12.1.

TABLE 12.1 net charge (in Hydrophathy Melting oxidizing Hydrophathy(GRAVY Temper- conditions) (GRAVY index) CDRs ature Anti- pH pH index)according to (C°) body pI 5.5 7.4 whole mAb IMGT Tm1 1204/ 9.05 32.9 7.4−0.401 −0.465 68 1205

Measurement of Kinetic Constants by Surface Plasmon Resonance

Human CD137 (R&D systems) was immobilized to the Biacore™ sensor chip,CMS, using conventional amine coupling. The tested antibodies andcontrols (serially diluted 1/2 10-0.63 nM) were analyzed for binding inHBS-P (GE, # BR-1003-68) at a flow rate of 30 μl/ml. The association wasfollowed for 5 minutes and the dissociation for 15 minutes. Regenerationwas performed twice using 10 mM Glycine pH1.7 for 30 seconds. Thekinetic parameters and the affinity constants were calculated using 1:1Langmuir model. As a representative example, the 1204/1205 anti-CD137antibody has an affinity in the low nanomolar range. See Table 12.2.

TABLE 12.2 Dissociation constant K_(D) (M) 1.1E−09 Association rateconstant k_(a) (1/Ms) 2.5E+05 Dissociation rate constant k_(d) (1/s)2.8E−04

Measurement by ELISA of Binding to Human CD137

Binding of CD137 mAb to recombinant human CD137 was determined bysandwich ELISA. Briefly, ELISA plates (Greiner #655074) were coated withrecombinant human CD137-Fc (R&D #838-4B) at 0.5 μg/ml, or alternatively0.05 μg/ml at 4° C. overnight or 37° C. for 1 hour. The plates werewashed three times with PBS+0.05% Tween 20 (PBST) and blocked withPBST+1% BSA. The CD137 antibodies to be tested were added in serialdilution series and the incubated for 1 h at room temperature prior towash with PBST. Binding was detected using HRP-conjugatedgoat-anti-human kappa light chain (AbD Serotec # STAR127P) and developedwith SuperSignal ELISA Pico Chemiluminescent substrate (Pierce #37069)measured using a Fluostar Optima. EC50 values of the various mAb weredetermined in 2-6 separate experiments.

As a representative example, the 1204/1205 anti-CD137 antibody has anEC50 value of approximately 0.3 nM when assessed by this method. SeeTable 12.3.

TABLE 12.3 Clone name Mean (nM) SD n 1204/1205 0.34 0.058 6 n = numberof data points, including separate experiments and different antibodybatches in same experiment

Measurement of Binding to Human or Cynomolgus CD137 Over-Expressed onCHO Cells

The extracellular part of human or cynomolgus CD137 was fused to thetransmembrane and intracellular part of hCD40 and cloned into pcDNA3.0.The vector was subsequently stably transfected into CHO cells.Expression of CD137 was confirmed by incubating with commercial CD137antibody (huCD137-PE, BD Biosciences #555956) for 30 min at 4° C.

For the assay, the transfected cells were incubated with the testantibodies and controls for at least 1 hr at 4° C. to saturate binding.In order to minimize antibody internalization, 0.05% sodium azide wasused in the incubation buffer and all work was performed on ice.Detection was achieved using an anti-hIgG-PE antibody (109-115-098,Jackson Immunoresearch laboratories) incubated for 30 min at 4° C.Directly after staining, the cells were fixed with a paraformaldehydesolution (10× concentrate BD CellFIX, BD biosciences #340181). Cellswere analyzed by flow cytometry using FACSVerse (BD Biosciences). Themedian fluorescence intensity (MFI) for each sample was determined andthe dose response data was analyzed using Graph Pad Prism. In order tofit MFI data in Graph Pad MFI data was normalized for each antibody,where 0% was defined as the lowest value and 100% was the highest valuein the dose titration for each antibody.

Binding to human and cynomolgus monkey CD137 was confirmed in twoseparate experiments for exemplary antibody 1204/1205. The bindingaffinities were very similar between cynomolgus monkey and human CD137(FIG. 14, Table 12.4)

TABLE 12.4 95% confidence intervals for the EC50 determined as anaverage from two experiments of normalized data. Binding to humanBinding to cyno Clone name CD137, EC50 (μg/mL) CD137, EC50 (μg/mL)1204/1205 0.23-0.39 0.11-0.16

Agonistic Activity of CD137 Antibodies in a Human T Cell Assay

Agonistic activity of CD137 mAb was evaluated in a T cell assay based onprimary human CD8⁺ T cells. Briefly, CD8⁺ T cells were separated fromhuman peripheral blood mononuclear cells by MACS separation (Miltenyi#130-096-495) according to the manufacturer's protocol. Cells wereincubated in 96-well microtiter plates (NuncThermo Scientific #268200),pre-coated with anti-CD3 mAb (clone OKT3, Affymetrix eBioscience#16-0037) and titrated concentrations of the CD137 mAb to be tested.Following 72 or 96 hour incubation, culture medium was harvested andIFN-γ levels were determined by ELISA (BD #555142). Each clone wasanalyzed in multiple donors and compared to a reference CD137 mAb111/112 and an isotype control (62/63).

Due to large intra-donor variations the stimulation index (SI, foldinduction by mAb compared to isotype control) was determined for eachsample and normalized to the stimulation index for the referenceantibody 111/112.

The exemplary 1204/1205 antibody induced activation of T cells that wascomparable or better compared to the reference antibody 111/112. Meannormalized SI±SD of 8 donors is presented in FIG. 15. Table 12.5indicates the absolute IFN-γ levels induced by CD137 stimulation.

TABLE 12.5 IFN-γ production levels induced by the various mAb Ab CloneMean IFN-γ Min IFN-γ Max IFN-γ No. of repeated name (pg/ml) (pg/ml)(pg/ml) experiments 62/63 2502 337 8526 13 111/112 42268 2256 136802 121204/1205 64430 13062 153136 8

Sequence Information

(SEQ ID NO: 207) VH CDR1 = GFTFSSYY (SEQ ID NO: 212) VH CDR2 = IGSYYGYT(SEQ ID NO: 217) VH CDR3 = ARAYYDYNYYYAYFDY (SEQ ID NO: 80)VL CDR1 = QSISSY (SEQ ID NO: 81) VL CDR2 = AAS (SEQ ID NO: 222)VL CDR3 = QQSVPHYPFT

TABLE 12.6 1205, light DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWY chain VL aaQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFT sequenceLTISSLQPEDFATYYCQQSVPHYPFTFGQGTKLEIK (SEQ ID NO: 177) 1205, lightGACATCCAGATGACCCAGTCTCCATCCTCCCTGAGC chain VL ntGCATCTGTAGGAGACCGCGTCACCATCACTTGCCGG sequenceGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCACGTTTCAGTGGCAGTGGAAGCGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTATTACTGTCAACAGTCTGTTCCGCACTACCCGTTCACTTTTGGCCAGGGGACCAAGCTGGAGATCAAA (SEQ ID NO: 178) 1204, heavyEVQLLESGGGLVQPGGSLRLSCAASGFTFSSYYMGW chain VH aaVRQAPGKGLEWVSGIGSYYGYTGYADSVKGRFTISR sequenceDNSKNTLYLQMNSLRAEDTAVYYCARAYYDYNYYYA YFDYWGQGTLVTVSS (SEQ ID NO: 179)1204, heavy GAGGTGCAGCTGTTGGAGAGCGGGGGAGGCTTGGTA chain VH ntCAGCCTGGGGGGTCCCTGCGCCTCTCCTGTGCAGCC sequenceAGCGGATTCACCTTTTCTTCTTACTACATGGGTTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTGGTTCTTACTACGGTTACACAGGTTATGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCCGTGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGCGTGCCGAGGACACGGCTGTATATTATTGTGCGCGCGCTTACTACGACTACAACTACTACTACGCTTACTTTGACTATTGGGGCCAGGGAACCCTGGTCACC GTCTCCTCA (SEQ ID NO: 180)

Example 13—Domain Mapping of CD137 Antibodies

The extracellular part of CD137 consists of four domains, which can befurther subdivided into two modules. Genes of CD137 human/mouse chimeraswere synthesized using standard laboratory techniques. The differentchimeras were designed by exchanging domains or modules of the humanCD137 with corresponding mouse CD137. The chimeras were designed basedon evaluation of the human and mouse sequences and 3D investigation ofhuman CD137. The synthesized genes were assigned project specific IDnumbers (see Table 13.1). The constructs were cloned into pcDNA3.1vector (Invitrogen) and transiently transfected into FreeStyle 293-Fcells (Invitrogen). The transfected cells were incubated with CD137antibodies and control antibodies, followed by incubation withanti-human IgG-PE (Jackson Immunoresearch) for detection and analyzedwith FACS Verse (BD Biosciences). Binding to the different chimericconstructs was calculated as relative MFI compared to the binding of theisotype control, followed by normalization to the full-length humanCD137 construct to minimize the effect of affinity differences betweenindividual antibodies.

The results for binding of exemplary antibody 1204/1205 to the differentconstructs are shown in Table 13.2. As indicated by these results,antibody 1204/1205 is mainly dependent on domain 2. In addition, someloss of binding is also seen for construct 1555, indicating an impact ofdomain 1 as well.

TABLE 13.1 CD137 constructs used for the domain mapping SequenceDescription 1550 (1407) Human CD137 with mouse domains 1, 2A and 2B (aa24-86) 1551 (1408) Human CD137 with mouse domains 2A, 2B and 3A (aa47-96) 1552 (1409) Human CD137 with mouse domains 2B, 3A and 3B (aa64-118) 1553 (1410) Human CD137 with mouse domains 3A, 3B and 4A (aa87-133) 1554 (1411) Human CD137 with mouse domains 3B, 4A and 4B (aa97-159) 1555 (1412) Human CD137 with mouse domains 1 and 4B and regionof unknown function (aa 24-46 and aa 139-186) 1030 Full length humanCD137

(wherein the number in parentheses identifies the same CD137 construct,but corresponds to an alternative clone numbering system used in thefigures)

TABLE 13.2 Binding of antibody 1204/1205 to the different CD137constructs measured by flow cytometry. Median fluorescence intensity(MFI) for mAb sample/isotype control, normalized to full-length humanCD137. Sequence Relative binding 1550 0.07 1551 0.11 1552 0.13 1553 0.851554 0.73 1555 0.28 1030 1

Bispecific Antibodies

Example 14—Agonistic Activity of Exemplary Bispecific Molecules In Vitro

Human CD3 positive T cells were purified from Ficoll separated PBMCs(obtained from leucocyte filters from the blood bank of the LundUniversity Hospital) using negative selection (Pan T cell Isolation Kit,human, Miltenyi, 130-096-535). 50 μl of anti-CD3 (clone: UCHT-1, BD,concentration: 1 μg/ml) diluted in PBS was coated to the surface ofnon-tissue culture treated, U-shaped 96-well plates (Nunc, VWR#738-0147) over night at 4° C. Bispecific anti-OX40/anti-CD137polypeptides were added in a serial dilution to the wells and comparedat the same molar concentrations as controls. Two different controlswere used for each bispecific molecule tested. The first control is anisotype control antibody specific for GFP (designated 1188-1187). Thesecond control is a mixture of the monospecific OX40 and CD137antibodies corresponding to the tested bispecific. After 72 h ofincubation in a moisture chamber at 37° C., 5% CO2, IL-2 levels weremeasured in the supernatant.

As shown in FIG. 16A-H, there is a dose-dependent effect of thebispecific molecules, which induce an increase in human T cellactivation (measured by an increase in IL-2 production) when cultured inplates coated with anti-CD3. The controls do not. FIG. 17 shows theresults of a similar assay when conducted at a fixed concentration forthe exemplary bispecific antibody 1164/1135-1204/1205 and controls (1nM). In this assay Human PBMC were obtained from leucocyte concentratesfrom the blood bank of the Lund University Hospital) using Ficolldensity separation. 50 μl of anti-CD3 (clone: UCHT-1, BD, concentration:1 μg/ml) diluted in PBS was coated to the surface of a non-tissuecultured treated, U-shaped 96-well plates (Nunc, VWR #738-0147) overnight at 4° C. The bispecific anti-OX40/anti-CD137 antibody1164/1135-1204/1205 was added soluble to the wells together with PBMCsand compared at the same molar concentration (1 nM) with an isotypecontrol 1188-1187 or its corresponding monospecific OX40 and CD137antibodies. After 48 h of incubation in a moisture chamber at 37° C., 5%C02, IL-2 levels were measured in the supernatants. The bispecificantibody induces significantly higher T cell activation than controlseven in a mixed population of different cells (PBMCs).

Since this assay represents an experimental model of a tumormicroenvironment where both OX40 and CD137 are relatively overexpressed,the results suggest that the tested bispecific molecules can be expectedto have a greater effect than monospecific antibodies in such amicroenvironment.

Example 15—Dual Binding to Both Targets of Exemplary BispecificMolecules

Measurement by ELISA

ELISA plates were coated with OX40-Fc (R&D systems, #3388_OX) (0.4μg/ml, 50 μl/well) over night at 4° C. The ELISA plates were washed withPBST and then blocked with PBST+2% BSA for 1 h at room temperature.After 3 washes with PBST, bispecific antibodies and controls were addedat different concentrations, from 50 nM to 6.4×10⁻⁴ nM and incubated for1 h at room temperature. The ELISA plates were washed and biotinylatedCD137-Fc at (1 μg/ml) was added and incubated on the plates for 1 h atroom temperature. The plates were washed three times with PBST andHRP-labelled streptavidin was used for detection of binding (1 hincubation at room temperature). The plates were washed 6 times withPBST and then SuperSignal Pico Luminescent was used as substrate andluminescence was measured using Fluostar Optima according to themanufacturers' protocols.

Binding to both targets was confirmed for all tested bispecificmolecules as is shown in FIG. 18. Each of the bispecific antibodies wasdetectable at 8×10⁻² nM. The bispecific antibodies also bind to bothtargets when the assay is reversed (coating CD137 and using solublebiotinylated OX40 for detection (data not shown).

Example 16—Selection of CD137 Antibodies from Alligator GOLD™ Libraryand Overview of Tested Clones

Phage display selections were performed using a human antibody (scFv)library, Alligator GOLD. Selections towards recombinant CD137 in solubleform, coated onto the surface of beads or tubes, or expressed on thesurface of CD137-transfected cells were performed. CTLA4-Fc and anirrelevant His-tagged protein were used as non-targets included inexcess in the selections. Prior to each selection round, the phagestocks were pre-selected towards biotinylated beriglobin, CTLA4-Fc,beads or CD137 negative cells to remove unspecific binders.

To identify specific binders from the phage selection, approximately4500 individual clones were screened in phage format using ELISA coatedwith either recombinant target (CD137-Fc) or non-target (CTLA4-Fc)protein, followed by confirmation as soluble scFv for some clones.Clones exhibiting specific binding to CD137 were sequenced and uniqueclones were produced as IgG for further characterization.

Five human CD137 antibodies with agonistic properties have beencharacterized and described herein (summarized in Table 16.1). Thetested antibodies are comparable to clinically active referenceantibodies in a functional T cell assay. Four different classes ofantibodies were identified based on epitope domain mapping studies. BothCD137 blocking and non-blocking antibodies were obtained.

TABLE 16.1 Summary of the CD137 antibodies Cynomolgus EC50 cell CD137binding (CHO T cell cross- Target transfectants) ka kd KD CD137L DomainAntibody agonist reactivity Specificity (μg/mL) (1/Ms) (1/s) (M) blockbinding 1204/1205 Yes Yes OK 0.23-0.39 3E+05 3E−04 1E−09 Yes 2 1214/1215Yes Yes OK 0.89-1.28 5E+04 3E−05 7E−10 No ND 1618/1619 Yes Yes OK0.11-0.19 1E+06 1E−04 1E−10 No 1 1620/1621 Yes Yes OK 0.20-0.42 4E+055E−04 1E−09 Yes 3B-4A 1626/1627 Yes Yes OK 0.38-0.67 2E+05 3E−04 1E−09No ND

Example 17—Characteristics of CD137 Antibodies—Binding to Human CD137Measured by ELISA

Binding of CD137 antibodies to recombinant human CD137 was determined bysandwich ELISA. Briefly, ELISA plates (Greiner #655074) coated withrecombinant human CD137-Fc (R&D #838-4B) were incubated with serialdilutions of the various CD137 antibodies to be investigated. CD137antibodies were detected using HRP-conjugated goat-anti-human kappalight chain (AbD Serotec # STAR127P) and developed with SuperSignalELISA Pico Chemiluminescent substrate (Pierce #37069). EC50 values ofthe various antibodies were determined in multiple separate experiments.

Two different reference antibodies have been used in this study,designated 1811/1812 and 1813/1814.

Reference antibody 1811/1812 is a CD137 agonist that binds to domain 1of CD137 and does not block the ligand. It binds to its target with highaffinity and specificity. An antibody with this sequence has beenevaluated in clinical trials.

Reference antibody 1813/1814 is and antibody that binds to domain 3-4 onCD137. It binds to its target with high affinity and specificity. Anantibody with this sequence has been evaluated in clinical trials.

The majority of the antibodies exhibit EC50 values in a similar range asthose of the reference antibodies, i.e. sub nM or low nM. Data issummarized in Table 17.1.

TABLE 17.1 EC50 values (nM) of Alligator-GOLD-derived CD137 antibodiesdetermined by ELISA for human CD137. Clone name Mean SD n 1811/1812 0.750.137 8 1813/1814 0.33 0.069 5 1204/1205 0.34 0.058 6 1214/1215 0.980.124 6 1618/1619 0.35 0.018 4 1620/1621 0.38 0.137 2 1626/1627 0.220.057 2 n = number of data points.

Example 18—Characteristics of CD137 Antibodies—Binding to Human andCynomolgus CD137 Measured by Flow Cytometry

Binding and EC50 to human and cynomolgus (Macaca fascicularis) CD137 wasdetermined using flow cytometry of CHO cells transfected with humanCD137, cynomolgus CD137 or empty vector. The extracellular part of humanor cynomolgus CD137 was fused to the transmembrane and intracellularpart of human CD40 and cloned into pcDNA3.0. The vector was subsequentlystably transfected into CHO cells. Expression of CD137 was confirmed byflow cytometry using CD137 antibody (human CD137-PE, BD Biosciences#555956) for 30 min at 4° C. CD137-transfected and emptyvector-transfected cells were incubated with CD137 antibodies for atleast 1 h at 4° C. to saturate the binding. In order to minimizeantibody internalization, 0.05% sodium azide was used in the incubationbuffer and all work was performed on ice. The CD137 antibodies weredetected using PE-conjugated anti-hIgG antibody (109-115-098, JacksonImmunoresearch laboratories), incubated for 30 min at 4° C. Directlyafter staining the cells were fixed with a paraformaldehyde solution(10× concentrate BD CellFIX, BD biosciences #340181). Cells wereanalyzed by flow cytometry using FACSVerse (BD Biosciences). The medianfluorescence intensity (MFI) for each sample was determined and the doseresponse data was analysed using Graph Pad Prism.

MFI data was normalized for each antibody, where 0% is defined as thelowest value and 100% is the highest value in the dose titration foreach antibody. EC50 and 95% confidence interval were calculated withGraph-Pad Prism based on data from the two experiments (non-linearregression (curve fit), constraints set to 0 and 100).

Binding to CHO-huCD137, CHO-cyCD137 and CHO-pcDNA was confirmed in twoseparate experiments (FIG. 20). All CD137 antibodies bind relativelywell to human CD137 with EC50 comparable with the two referenceantibodies 1181/1812 and 1813/1814. All CD137 antibodies tested bindwell to cynomolgus CD137, except for reference antibody 1811/1812 whichdoes not bind at all and clone 1620/1621 which binds weakly and does notreach a complete saturation. The binding pattern has also been confirmedon stimulated primary cynomolgus PBL.

The EC50 determination is presented as 95% confidence intervals for eachCD137 antibody tested in order to include the inter and intra assayvariations (Table 18.1)

TABLE 18.1 95% confidence intervals for the EC50 of each CD137 antibodydetermined as an average from two experiments of normalized data.Binding to human Binding to cyno Ratio, Clone name CD137, EC50 (μg/mL)CD137, EC50 (μg/mL) cyno:human 1204/1205 0.23-0.39 0.11-0.16 0.431214/1215 0.89-1.28 0.41-0.80 0.54 1618/1619 0.11-0.19 0.086-0.15  0.771620/1621 0.20-0.42  3-5* 14*   1626/1627 0.38-0.67 0.16-0.27 0.41

Example 19—Characteristics of CD137 Antibodies—Affinity Measured byBiacore

Human CD137 (R&D systems) was immobilized to the Biacore™ sensorchip,CMS, using conventional amine coupling. The tested antibody and control(serially diluted 1/2 10-0.63 nM) were analyzed for binding in HBS-P(GE, # BR-1003-68) at a flow rate of 30 μl/ml. The association wasfollowed for 5 minutes and the dissociation for 15 minutes. Regenerationwas performed twice using 10 mM Glycine pH 1.7 for 30 seconds. Thekinetic parameters and the affinity constants were calculated using 1:1Langmuir model.

Results and Conclusions

The affinities of the antibodies were in the nanomolar to sub-nanomolarrange (Table 19.1) measured using bivalent antibodies flowed over CD137coated on the chip surface.

TABLE 19.1 Kinetic parameters measured by surface plasmon resonanceSample ka (1/Ms) kd (1/s) KD (M) 1204 2.54E+05 2.80E−04 1.10E−09 12144.54E+04 3.17E−05 6.99E−10 1618 1.02E+06 1.10E−04 1.07E−10 1620 3.92E+055.19E−04 1.32E−09 1626 2.32E+05 2.94E−04 1.27E−09

Example 20—Characteristics of CD137 Antibodies—Target Specificity of theCD137 Antibodies

Determination of unspecific binding of CD137 antibodies to other TNFRsuperfamily members (CD40 and OX40) was evaluated to detect potentialpropensity to cross react to non-target proteins.

ELISA plates (Greiner #655074) were coated with 50 μl/well ofrecombinant human OX40 (R&D #1493-CD), CD40-Fc (Ancell #504-820) orCD137 (R&D #838-4B) diluted to a final concentration of 0.5 μg/ml in PBSfor 1 h at 37° C. or overnight at 4° C. Plates were washed withPBS+0.05% TWEEN20 (PBST), followed by block with PBST+1% bovine serumalbumin (BSA). Antibody samples were prepared as serial 1/10 dilutionsfrom 10-0.01 μg/ml in PBST+1% BSA and incubated for 1 h in roomtemperature, followed by detection using a horse radishperoxidase-conjugated anti-human kappa light chain antibody (AbD Serotec# STAR127P) and developed using SuperSignal ELISA Pico Chemiluminescentsubstrate (Pierce ThermoScientific #37069).

Results and Conclusions

TABLE 20.1 Summary of CD137 antibody unspecific binding to OX40 and CD40pAb Binding to OX40 and CD40 1204/1205 No 1214/1215 No 1618/1619 No1620/1621 No 1626/1627 No

No binding to OX40 or CD40 of the CD137 antibodies was detected. Anoverview of antibodies analyzed, and results from the two experiments isshown in Table 20.1.

Further, binding to primary PBL from multiple blood donors was tested.The binding to PBL was similar to Reference antibodies. No relevantunspecific binding to non-target proteins was detected.

Example 21—Characteristics of CD137 Antibodies—Domain Mapping ofAntibodies Binding to CD137

The ability of each antibody to bind to a panel of human/mouse CD137chimeras expressed on the surface of transfected cells was analyzed byflow cytometry.

The chimeras were designed by exchanging domains or modules of the humanCD137 with the corresponding mouse domain (FIG. 21). Genes of CD137human/mouse chimeras were synthesized (GenScript) and constructs clonedinto pcDNA3.1 vector (Invitrogen) and transiently transfected intoFreeStyle 293-F cells (Invitrogen). The transfected cells were incubatedwith CD137 antibodies and control antibodies, followed by incubationwith anti-human IgG-PE (Jackson Immunoresearch) for detection andanalyzed with FACS Verse (BD Biosciences). Binding to the differentchimeric constructs was calculated as relative MFI compared to thebinding of the isotype control, followed by normalization to thefull-length human CD137 construct to minimize the effect of affinitydifferences between individual antibodies.

Results and Conclusions

6 binding patterns can be observed as described below. Data issummarized in Table 21.1.

Pattern A

Antibody 1618/1619 is dependent on domain 1.

Pattern B

Antibody 1204/1205 is mainly dependent on domain 2. In addition, someloss of binding is also seen for construct 1555, indicating an impact ofdomain 1 as well.

Pattern C

Antibody 1620/1621 appears to be mainly dependent on domains 3B-4A.However, loss of binding is seen for all constructs, making this patternquite similar to pattern D.

Pattern D

For antibodies 1214/1215 and 1626/1627, no clear dependence onparticular CD137 domains could be demonstrated. Instead, theseantibodies exhibited extensive loss of binding for all chimeras.

TABLE 21.1 Domain mapping of CD137 antibodies. The table displaysrelative binding to different CD137 constructs in which part of thehuman CD137 sequence has been exchanged to the murine CD137 sequence.Median fluorescence intensity (MFI) for antibody sample/isotype control,normalized to full-length human CD137. Group A B C D Domain 1 2 3B-4AUnclear Clone 1214/1215 1618/1619 1204/1205 1620/1621 1626/1627 Exp 1Exp 2 1550 0.11 0.07 0.17 0.10 0.06 0.14 (1407) 1551 0.67 0.11 0.33 0.110.07 0.15 (1408) 1552 1.20 0.13 0.18 0.11 0.32 0.13 (1409) 1553 1.240.85 0.17 0.14 0.41 0.15 (1410) 1554 1.01 0.73 0.17 0.12 0.26 0.15(1411) 1555 0.12 0.28 0.32 0.29 0.30 0.45 (1412)  1030* 1 1 1 1 1 1*Full-length CD137 (wherein the number in parentheses identifies thesame CD137 construct, but corresponds to an alternative clone numberingsystem used in the figures)

Example 22—Characteristics of CD137 Antibodies—CD137 Ligand Blocking

The aim was to determine if the CD137 antibodies block the CD137 ligand(CD137L) binding.

In the previous domain mapping experiment the CD137 antibodies weredivided in different groups based on their binding to similar subdomainsof the CD137 antigen. If the CD137 antibodies bind to epitopes close tothe ligand binding region, binding to the antigen can lead to partly ortotal block of ligand biding. Binding close to the CD137 ligand bindingepitope may also affect the ligand binding due to steric hindrance orconformational changes of the CD137 ligand binding epitope. All CD137antibodies were titrated against a fixed concentration of CD137L forevaluation of ligand blocking properties.

CHO-cells transfected with human CD137 were used for the ligandcompetition. The extracellular part of human CD137 was fused to thetransmembrane and intracellular part of hCD40 and cloned into pcDNA3.0.The vector was subsequently stably transfected into CHO cells. Theexpression of CD137 was confirmed by staining with commercial antibodytargeting CD137.

The CHO-huCD137 were pre-incubated with CD137 monoclonal antibodies,titrating down from a predetermined saturating concentration (25, 2.5and 0.25 μg/ml), for 1 h at +4 C before the addition of CD137 ligand ata concentration at EC50. After co-incubation for another 30 min at +4 C,the cells were washed and bound CD137 ligand was detected withanti-FLAG-APC (Cell signaling technology). Before analyzation the cellswere fixed with paraformaldehyde (10× concentrate BD CellFIX, BDbiosciences). Analyzation was performed with FACSverse and the MFI(Median Fluorescence Intensity) was calculated with FlowJo software.

Results and Conclusions

The CD137L blocking experiment was performed two times. It can beconcluded that not all CD137 mAbs tested were blocking the CD137 ligandbinding (Table 22.1, FIG. 22). CD137 mAbs belonging to group B and C(1204 and 1620), binding to domain 2B-4A, were blocking the CD137L.Antibody 1814 has been reported to block the CD137L, however, this couldnot be verified in our CD137L blocking assay. 1812 and 1618, belongingto group A which bound to domain 1, did not block CD137 ligand, butinstead synergistically increased the CD137L binding. Antibodies 1626and 1214, did not block the CD137L in the two experiments performed.

TABLE 22.1 Maximal CD137 ligand competition of the CD137 antibodies,mean out of two experiments Group (domain mapping) CD137 mAb CD137L, maxinhib. A 1618/1619 −22%   B 1204/1205 50% C 1620/1621 56% D 1626/162718% D 1214/1215 11%

Example 23—Characteristics of CD137 Antibodies—Competition ELISA

By competing each CD137 antibody with each another, it is possible todetermine antibodies binding to similar epitopes based on their blockingpattern. The competition ELISA is performed by co-incubatingbiotinylated CD137 antibodies with non-biotinylated CD137 antibodieswhen binding to coated CD137-Fc. Competition is defined as loss ofsignal from the biotinylated CD137 antibody. Low competition valuescould either be due to no competition between the antibodies or bindingkinetics of the antibodies. Binding of one antibody could also lead tosteric hindrance or conformational changes when binding the antigenwhich affects the binding of the other CD137 antibody.

CD137 antibodies were biotinylated (EZ-link NHS-LC-Biotin, ThermoFisher)and intact binding properties to CD137-Fc was verified with ELISA bycomparing EC50 between biotinylated and non-biotinylated anti-CD137mAbs. Non-biotinylated anti-CD137 (anti-CD137) was pre-incubated toCD137-Fc at concentrations 30 times higher than the determined EC50 for0.5 h. Without washing, anti-CD137-bio was added and co-incubated foranother 1 h. The binding of anti-CD137-bio was detected withStreptavidin-HRP (Pierce). Competition was calculated as the relativenumber by dividing the binding measured to other antibodies relative toits maximum competition (competing with itself). The relative valuesobtained were normalized against the maximum blocking capacity (Table23.1).

TABLE 23.1 Summary of CD137 antibody competition ELISA from twoexperiments Values are presented as % competition with CD137-bio Groupcomp ELISA X Y Group (domain mapping) A B C D 1618/1619 1204/12051620/1621 1626/1627 1214/1215 1812-bio 100 5 4 0 4 1814-bio 21 74 61 5799 1214-bio 6 92 80 77 99 1618-bio 88 3 10 16 9 1620-bio 7 93 82 79 1001626-bio 24 100 97 100 99 1204-bio 28 88 66 66 97

Results and Conclusions

The competition ELISA was performed twice. In both experiments, severalof the CD137 mAbs did not fully compete with itself. When normalizingthe relative competition values for each antibody a competition patternemerged (Table 23.1). The antibodies 1812 and 1618 that belongs todomain mapping group A, displayed a unique pattern in the competitionELISA (group X). The other CD137 antibodies analyzed had asimilarblocking pattern (group Y). Differences in binding kinetics may explainsome of the minor variations in the binding patterns among theantibodies of group Y, although it cannot be excluded that the smallvariations within group Y reflects actual differences in the epitopebinding.

Example 24—Characteristics of CD137 Antibodies—In Vitro Efficacy ofCD137 Antibodies

The aim was to identify CD137 antibodies with agonistic activity.

Agonistic activity of CD137 antibodies was evaluated in a T cell assaybased on primary human CD8⁺ T cells. Briefly, CD8⁺ T cells wereseparated from human peripheral blood mononuclear cells by MACSseparation (Miltenyi #130-096-495) according to the manufacturer'sprotocol. Cells were incubated in 96-well microtiter plates (NuncThermoScientific #268200), pre-coated with anti-CD3 antibody (clone OKT3,Affymetrix eBioscience #16-0037) and titrated concentrations of theCD137 antibody to be tested. Following 72 or 96 hour incubation, culturemedium was harvested and IFN-γ levels were determined by ELISA (BD#555142).

Each clone was analyzed in at least 6 donors and compared to thereference CD137 antibody 1811/1812 and the negative control antibody.

Due to large intra-donor variations the stimulation index (SI, foldinduction by antibody compared to negative control) was determined foreach sample and normalized to the stimulation index for the referenceantibody 1811/1812.

Results and Conclusions

Several clones with efficacy comparable to the reference 1811/1812 wereidentified (see FIG. 23).

Table 24.1 indicates the absolute IFN-γ levels induced by CD137stimulation. However, all antibodies were not analyzed head-to-head inall donors, and the normalized SI is more relevant for comparison of theefficacy. The antibodies were evaluated in an IgG1 format, and theefficacy was measured using antibodies coated to the surface of thewells, which may influence the efficacy.

TABLE 24.1 IFN-γ production levels induced by the various antibodies.Mean IFN-γ Min IFN-γ Max IFN-γ Clone name (pg/ml) (pg/ml) (pg/ml) n CtrlIgG 2502 337 8526 13 1204/1205 64430 13062 153136 8 1214/1215 51836 8208122163 8 1618/1619 33604 7380 111196 8 1620/1621 52448 7727 123127 81626/1627 15097 4159 32163 6

Bispecific Antibodies

Example 25—Bispecific Antibodies Targeting CD137 and CTLA4—Binding toBoth Targetsin Dual ELISA

Aim: To evaluate and confirm the ability of the bispecific antibodies tobind to both targets using ELISA.

Material and Methods

ELISA plates were coated with rhCD137-Fc (rh4-1BB, 0.5 μg/ml) over nightat 4° C. Bispecific antibodies were added in dilutions and detected byaddition of biotinylated CTLA-4 (1 μg/ml) followed by HRP-labelledstreptavidin (0.167 μg/ml). SuperSignal Pico Luminescent was used assubstrate and luminescence was measured using Fluostar Optima.

Results

The bispecific antibodies could be detected at approximately 0.1 nM, anddisplayed EC50 values of approximately 0.5-1 nM in this assay (see FIG.24). The results show that the bispecific CD137-CTLA-4 antibodies canbind to both targets simultaneously

Example 26—Bispecific Antibodies Targeting CD137 and CTLA4—ActivationAbility in the Context of Cells Expressing CTLA-4

Aim

To determine the ability to activate immune cells in the context ofcells expressing CTLA-4. The aim is to achieve higher activation(efficacy and potency) when CTLA-4 is present.

Material and Methods

Human CD8 positive T cells were obtained using negative selection(Miltenyi, human CD8+ T cell Isolation Kit, 130-096-495) of PBMC fromleucocyte filters obtained from the blood bank (Lund UniversityHospital). CTLA-4 (Orencia, 2.5 μg/ml) and anti-CD3 (OKT-3, 3 ug/ml) wascoated to the surface of a 96 well U-shaped culture plate (Nunc, VWR,#738-0147, non-tissue culture treated) over night at 4° C. Bispecificantibodies and controls were added to the wells. The IFNγ levels in thesupernatant was measured after 96 h of incubation in a moisture chamberat 37° C., 5% CO₂.

Results

The results (FIG. 25, FIG. 26) show that the bispecific antibodiesactivate T cells only when cultured on plates coated with CTLA-4. The invitro assay represents an experimental model of the situation in thetumor microenvironment where CTLA-4 is relatively overexpressed. Theresults thus implicate that the bispecific antibodies have an increasedeffect in an environment with high levels of CTLA-4, e.g. in the tumor.

Example 27—Synergism in Induction of ADCC by Exemplary BispecificAntibodies Targeting CTLA4 and OX40 (Compared to Effect of MonospecificAntibodies, Alone or in Combination)

Materials and Methods

Assessment of antibody-dependent cell-mediated cytotoxicity (ADCC)

Jurkat cells engineered to stably express FcγRIIIa receptor (V158variant) and an NFAT response element driving expression of fireflyluciferase (Promega Corporation) were used as effector cells in theassessment of ADCC. Antibodies were titrated in duplicate wells in a96-well opaque luminescence plate, and effector cells and target cellsexpressing both OX40 and CTLA4 were added in a ratio of 5:1. After 6 hincubation in a 37° C., 95% O₂ humidified incubator, luciferase assaysubstrate (Promega Corporation) was added to all wells including mediumcontrol wells (for blank subtraction), and luminescence was detected ona FLUOstar Optima microplate reader (BMG LabTech). Fold-induced ADCC wascalculated as: (target lysis−blank)/(spontaneous lysis−blank). Topvalues were calculated based on log(agonist) vs. response (threeparameters) curve fit using Prism 6.0 (Graphpad, La Jolla, Calif., USA).

Antibodies

-   -   “1166/1167”=monospecific OX40 antibody    -   “Control IgG with CTLA-4 binding part”=monospecific CTLA4        binding domain fused to an IgG protein    -   “1166/1261”=exemplary bispecific antibody targeting OX40 and        CTLA4 (containing the identical OX40 and CTLA-4 binding part as        the monospecific binders described above).    -   “Ctrl IgG”=negative isotype control

Results

Exemplary Bispecific Antibody 1166/1261 Exhibits Superior Induction ofADCC

As shown in FIG. 27, detectable levels of ADCC were induced by alltested components. The negative isotype control did not induce any ADCC(data not shown). Most notably, the bispecific 1166/1261 antibodyinduced ˜123-fold ADCC compared to control. The monospecific OX40antibody (1166/1167) induced ˜29-fold and the monospecific CTLA-4binding domain (62/376) induced ˜10-fold ADCC, whereas the mixture ofthe two monospecific components (1166/1167+62/376) induced ˜31-foldADCC.

There is thus an unexpected and marked synergy obtained by thebispecific molecule binding to OX40 and CTLA-4.

Example 28—Bispecific Antibodies Targeting OX40 and CTLA4—SpecificBinding to Cells Expressing Both OX40 and CTLA4

Background

The aim of this study was to determine the binding efficacy and EC50 of1166/1261 and the corresponding monospecific binding entities to cellsexpressing both OX40 and CTLA4 using flow cytometry. The bispecificantibody is designed to bind both OX40 and CTLA4 simultaneously. Forthis purpose, we used transfected CHO cells with a stable expression ofour targets. CHO P4 cells have a high expression level of both OX40 andCTLA4.

Methods and Results

Double-transfected CHO cells expressing both OX40 and CTLA4 wereoriginally sorted by FACS (Beckton Dickinson) into a cell poolexpressing high levels of both targets (denoted CHO P4). Targetexpression was kept stable by culturing the cells under selectionpressure of geneticine and zeocine. Untransfected CHO wild-type cellswere used as controls.

Cells were stained with decreasing concentrations of 1166/1261 (anexemplary bispecific antibody targeting OX40 and CTLA4), or the twomonospecific binders 1166/1167 (OX40 specific monoclonal antibody) andControl IgG with CTLA-4 binding part (monospecific CTLA4 binding IgGfusion protein) (200 nM-0,0034 nM), followed by PE-conjugated anti-humanIgG. Fluorescence was detected using a FACSverse instrument, and theacquisition was analysed using FlowJo software. The median fluorescentintensity (MFI) was determined for each staining.

Binding efficacy curves for CHO P4 are presented in FIG. 28 (onerepresentative experiment out of three). 1166/1261 binds to cells withhigh expression of OX40 and CTLA4 better than 1166/1167 or the ControlIgG with a CTLA-4 binding entity. This is probably an additive effect of1166/1261 being able to bind two targets simultaneously.

Example 29—Bispecific Antibodies Targeting OX40 and CTLA4—Dual Bindingof Cells Expressing OX40 and CTLA4 Measured by Flow Cytometry

Aim

Measure simultaneous binding by 1166/1261 to both OX40 and CTLA4over-expressed on cells by measuring the number of aggregated cellsusing flow cytometry.

Materials and Methods

CHO-OX40 cells and HEK-CTLA4 cells were intracellularly stained with thefluorescent dyes PKH-67 (green fluorescent dye) respectively PKH-26 (redfluorescent dye) (Sigma-Aldrich). After verifying homogenously stainedcell population, the cells were mixed and incubated with either1166/1261 (an exemplary bispecific antibody targeting OX40 and CTLA4) ora combination of the two monoclonal antibodies 1166/1167 (a monospecificanti-OX40 antibody) and a control IgG comprising a CTLA4-binding domain.After staining the cells were immediately fixed and the number ofaggregated, double-positive cells were quantified using FACS-verse (BDbiosciences). Data analyses and non-linear regression was performedusing Graph Pad Prism v6.

Results and Conclusions

Exemplary bispecific antibody 1166/1261 increases the number ofaggregated cells with increasing concentration (FIG. 29) (onerepresentative experiment out of two).

Example 30—Bispecific Antibodies Targeting OX40 andCTLA4—Pharmacokinetics in Mice

Material and Methods

Antibodies

-   -   1166/1261 (an exemplary bispecific antibody targeting OX40 and        CTLA4)    -   1166/1167 (a monospecific control antibody targeting OX40)

In Vivo Studies

Female C57BL/6 (7-8w) mice from Taconic's Denmark were used in theexperiments. All experiments were done by approval of Malmö/Lund ethicalcommittee.

The mice were injected intraperitoneally with 100 μg of each antibodyand blood was drawn either via vena saphena or at termination via venacava into heparinized tubes after 0 h, 1 h, 4 h, 8 h, 24 h, 72 h andafter 1 week. 3 mice were used for each time-point. Blood was spun at2500 rpm for 30 min and plasma was frozen to −80 C° for furtheranalysis.

Assays for Determination of 1166/1261 and 1166/1167 Levels in Plasma

Two different assays were used. A single target ELISA (ELISA1) and adual ELISA (ELISA2). Briefly the assays consisted of the followingsteps. White high-binding flat-bottom, LIA plates (Greiner Bio-One,Austria) were coated over night with 0.8 μg/mL humanOX40-Fc (RnDSystems, MN, USA). After washing with Washing buffer (phosphatase buffersaline supplemented with 0.05% Tween 20 (PBST), Medicago, Sweden) thewells were blocked using PBST with 2% bovine serum albumin (BSA) (Merck,Germany) for 1 hour at ambient room temperature (ART) with shaking andwashed again before plasma samples diluted 1:200 and 1:5000 in assaybuffer (PBST+0.5% BSA) together with calibration curve samples(1166/1261, conc. 6-0.0012 μg/mL) were added. After incubation at ARTfor 1 h with shaking and subsequent washing, secondary reagent wasadded, consisting of either human anti-kappa-antibody horse radishperoxidase conjugated (HRP) (AbD Serotec, UK) for the single targetELISA or biotinylated human CTLA-4-Fc (Orencia) at 1 μg/mL followed bystreptavidin-HRP (Thermo Fisher Scientific, MN, USA) according to themanufacturer's instructions for the dual ELISA. Signal was obtainedusing HRP substrate SuperSignal Pico Luminescence (Thermo FisherScientific). Luminescence measurements were collected after 10 minutesincubation in darkness with shaking using a Flurostar Optima (MBGLabtech, Germany). The data was analyzed by using GraphPad Prismprogram.

Results

Samples collected at the different time points after injection with1166/1261 and 1166/1167 were analyzed with only single target ELISA orsingle target and dual ELISA for determination of the plasma levels of1166/1261 and 1166/1167 respectively. The results show that the levelsof 1166/1261 and 1166/1167 in plasma increased around the first 4 hoursafter peritoneal injection and then reduced (FIG. 30 upper panel).Detectable levels of both 1166/1261 and 1166/1167 are present in plasmaafter one week (FIG. 30 middle and lower panels).

The levels of 1166/1261 in plasma are similar to the levels obtained forthe monoclonal antibody 1166/1167 indicating that 1166/1261 exhibits agood half-life in vivo, comparable to that of an equivalent monospecificanti-OX40 antibody.

REFERENCES

-   Hemerle T., Wulhfard S., Neri D., (2012) A critical evaluation of    the tumor-targeting properties of bispecific antibodies based on    quantitative biodistribution data. Protein Engineering and Design,    25, pp 851-854.

Example 31—Bispecific Antibodies Targeting OX40 and CTLA4—In VivoAnti-Tumor Effect in HT-29 Colon Cancer Model

Summary

The anti-tumor effect of 1166/1261 (an exemplary bispecific antibodytargeting OX40 and CTLA4) was investigated using hPBMC humanizedimmunodefiecient mice and subcutaneous tumor models of HT-29 coloncarcinoma.

1166-1261 demonstrated statistically significant tumor volumeinhibition.

Material and Methods

Female SCID-Beige mice (6-9w) from Taconic's Denmark were used in theexperiments. All experiments were done by approval of Malmö/Lund ethicalcommittee.

HT-29 colon cancer were obtained from ATCC and cultivated according toATCC recommendations. The HT-29 cell line growing in log phase wasinjected subcutaneously (4×10⁶ cells in 100-200 μL at day 0 (DO)). HumanPBMC (7×10⁶ in 200 μL) isolated from leukocyte concentrates was injectedintraperitoneally at the same day. Intraperitoneal treatments (667 pmol)were done at days 6, 13, and 20.

Leukocyte concentrates were obtained from Lund University Hospital.

Tumor was measured with a caliper in width, length and height of whichthe tumor volume calculated (w/2×l/2×h/2×pi×(4/3)). The animals wereterminated before the tumor volume reached 2 cm³, at wounding, oraffected health of the mice.

The data were analyzed by by Mann-Whitney test using the GraphPad Prismprogram. Responder donors were considered those donors that wereresponsive to the reference antibody 1874. Minimum of 10% average tumorinhibition during the exponential tumor growth period was considered asa response.

Results

Pooled data from mice engrafted with responder donors (4 donors from twoseparate experiments) demonstrated statistically significant anti-tumorefficacy at days 12-16 in the form of inhibition of tumor growth whentreated with the 1166/1261 antibody (p=0.0469 to p=0.0074, Mann-Whitneynon parametric, 2-tail) in comparison to the vehicle group (ZZ). Thepercentage of tumor volume inhibition ranged from 22-36% with 1166/1261between days 10 and 21 (see FIG. 31 and Table 31.1).

In conclusion, the anti-tumor effect of 1166/1261 was investigated usinghPBMC humanized immunodeficient mice and subcutaneous tumor models ofHT-29 colon carcinoma. 1166/1261 demonstrated statistically significanttumor volume inhibition.

TABLE 31.1 Statistical analysis and percent tumor inhibition Day afterTumor growth p-value tumor inhibition (tumor volume) (Mann- inoculationcompared to vehicle (%) Whitney 2-tail) D10 22.8 0.1298 D12 35.4 0.0315D14 35.9 0.0074 D16 31.5 0.0469 D19 30.8 0.1059 D21 22.1 0.1067

Example 32—Bispecific Antibodies Targeting OX40 and CTLA4—In VivoAnti-Tumor Effect in Raji Lymphoma Model

Summary

The anti-tumor effect of 1166/1261 (an exemplary bispecific antibodytargeting OX40 and CTLA4) was investigated using hPBMC humanizedimmunodeficient mice and subcutaneous tumor models of Raji B-celllymphoma.

1166/1261 demonstrated statistically significant tumor volumeinhibition.

Material and Methods

Female SCID-Beige mice (6-9w) from Taconic's Denmark were used in theexperiments. All experiments were done by approval of Malmö/Lund ethicalcommittee.

Raji B-cell lymphoma was obtained from ATCC and cultivated according toATCC recommendations. The Raji cell line growing in log phase wasinjected subcutaneously (10×10⁶ cells) together with human PBMC (10×10⁶in 200 μL), isolated from buffy coats. Intraperitoneal treatments (667pmol) were done at days 0, 7, and 14.

Buffy coats were obtained from Kalmar University Hospital.

Tumor size was measured with a caliper in width, length and height ofwhich the tumor volume calculated (w/2×l/2×h/2×pi×(4/3)). The animalswere terminated before the tumor volume reached 2 cm³, at wounding, oraffected health of the mice.

The data were analyzed by Mann-Whitney test using the GraphPad Prismprogram. Responder donors were considered those donors that wereresponsive to the reference antibody 1874. Minimum of 10% average tumorinhibition during the exponential tumor growth period was considered asa response.

Results and Conclusions

Pooled data from experimental groups with responding donors, thebispecific 1166/1261 antibody demonstrated statistically significantanti-tumor efficacy at days 14 and 21 (p=0.0068 and p=0,0288,Mann-Whitney, 2-tail) in comparison to the vehicle (Table 32.1).

TABLE 32.1 Statistical analysis and percent tumor inhibition Tumor TumorTumor growth volume in volume in inhibition (tumor p-value Day aftervehicle- animals volume) (Mann- tumor treated treated with compared tovehicle Whitney inoculation animals 1166/1261 (%) 2-tail) D10 14.2 13.86.1 0.6842 D12 35.7 21.5 39.9 0.0603 D14 61.8 33.6 45.7 0.0068 D17 105.376.0 27.8 0.3527 D19 205.1 133.3 35 0.0524 D21 314.8 187.0 40.6 0.0288D24 467.5 299.9 37.2 0.054 D26 529.7 360.1 32 0.063

1. A bispecific polypeptide comprising a first binding domain,designated B1, which is capable of specifically binding to a first Tcell target, and a second binding domain, designated B2, which iscapable of specifically binding to a second T cell target, wherein thefirst and second T cell targets are different targets; wherein the firstT cell target is OX40; wherein B1 is an antibody or antigen-bindingfragment thereof specific for OX40; wherein B1 comprises a heavy chainvariable region amino acid sequence comprising SEQ ID NO: 91, and the B1comprises a light chain variable region amino acid sequence comprisingSEQ ID NO: 89; wherein the second T cell target is CTLA-4; wherein B2 isa polypeptide binding domain specific for CTLA-4; wherein B2 comprisesan amino acid sequence comprising SEQ ID NO: 17 wherein the polypeptidecomprises at least one polypeptide chain arranged according to theformulae: a light chain of B1-(X)n-B2, written in the direction N-C; andwherein X is a linker and n is 0 or
 1. 2. The polypeptide according toclaim 1 wherein the antigen-binding fragment is selected from the groupconsisting of: an Fv fragment, a single chain Fv fragment, adisulphide-bonded Fv fragment, a Fab-like fragment, a Fab fragment, aFab′ fragment, a F(ab)₂ fragment, and domain antibodies.
 3. Thepolypeptide according to claim 1, wherein B1 comprises a human Fc regionor a variant of a said region, where the region is an IgG1, IgG2, IgG3or IgG4 region.
 4. The polypeptide according to claim 1, wherein thepolypeptide is capable of inducing antibody dependent cell cytotoxicity(ADCC), antibody-dependent cellular phagocytosis (ADCP),complement-dependent cytotoxicity (CDC), apoptosis, and/or inducingtumour immunity.
 5. The polypeptide according to claim 1 in which theCTLA-4 specifically bound by the polypeptide is primate or murineCTLA-4, and/or wherein the OX40 specifically bound by the polypeptide isprimate OX40.
 6. The polypeptide according to claim 1, wherein X is apeptide with the amino acid sequence SGGGGSGGGGS (SEQ ID NO: 47),SGGGGSGGGGSAP (SEQ ID NO: 48), NFSQP (SEQ ID NO:49), KRTVA (SEQ ID NO:50), GGGGSGGGGSGGGGS (SEQ ID NO: 144) or (SG)m, where m=1 to
 7. 7. Thepolypeptide according to claim 1, which binds to human OX40 with a Kd ofless than 50×10⁻¹⁰M, 25×10⁻¹⁰M, or 20×10⁻¹⁰M and/or which binds to humanCTLA-4 with a Kd value which is less than 60×10⁻⁹M, 25×10⁻⁹M, or10×10⁻⁹M.
 8. The polypeptide according to claim 1, which induces anincrease in the activity of an effector T cell, optionally wherein saidincrease is at least 1.5 fold, 4.5 fold or 7 fold higher than theincrease in activity of an effector T cell induced by a combination ofB1 and B2 administered to the T cell as separate molecules.
 9. Thepolypeptide according to claim 8, wherein said increase in T cellactivity is an increase in proliferation and/or IL-2 production by the Tcell.
 10. The polypeptide according to claim 1, wherein B1 exhibits atleast one of the following functional characteristics when presentindependently of B2: I. binding to human OX40 with a K_(D) value whichis less than 10×10⁻¹⁰M; II. does not bind to murine OX40; and III. doesnot bind to other human TNFR superfamily members.
 11. A method oftreating or preventing a disease or condition in an individual, themethod comprising administering to an individual a bispecificpolypeptide according to claim
 1. 12. The method according to claim 11wherein the disease or condition is cancer and optionally wherein theindividual is human.
 13. The method according to claim 11, wherein themethod comprises administering the bispecific antibody systemically orlocally, such as at the site of a tumour or into a tumour draining lymphnode.
 14. The method according to claim 11 wherein the cancer isprostate cancer, breast cancer, colorectal cancer, pancreatic cancer,ovarian cancer, lung cancer, cervical cancer, rhabdomyosarcoma,neuroblastoma, multiple myeloma, leukemia, acute lymphoblastic leukemia,melanoma, bladder cancer, gastric cancer, head and neck cancer, livercancer, skin cancer, lymphoma or glioblastoma.
 15. A compositioncomprising a bispecific polypeptide according to claim 1 and at leastone pharmaceutically acceptable diluent or carrier.
 16. The polypeptideaccording to claim 1 conjugated to an additional therapeutic moiety.